Compositions and methods for the detection, diagnosis and therapy of hematological malignancies

ABSTRACT

Disclosed are methods and compositions for the detection, diagnosis, prognosis, and therapy of hematological malignancies, and in particular, B cell leukemias, lymphomas and multiple myelomas. Disclosed are compositions, methods and kits for eliciting immune and T cell responses to specific malignancy-related antigenic polypeptides and antigenic polypeptide fragments thereof in an animal. Also disclosed are compositions and methods for use in the identification of cells and biological samples containing one or more hematological malignancy-related compositions, and methods for the detection and diagnosis of such diseases and affected cell types. Also disclosed are diagnostic and therapeutic kits, as well as methods for the diagnosis, therapy and/or prevention of a variety of leukemias and lymphomas.

CROSS-REFERENCES TO RELATED APPLICATIONS

This application is a continuation in part of the U.S. patentapplication Ser. No. 10/154,884, filed May 23, 2002, which is acontinuation in part of the U.S. patent application Ser. No. 10/040,862,filed Nov. 6, 2001, Attorney Docket No. 014058-013520US, entitledCOMPOSITIONS AND METHODS FOR THE DETECTION, DIAGNOSIS AND THERAPY OFHEMATOLOGICAL MALIGNANCIES, the entire specification, claims, sequencesand figures of each of which is specifically incorporated herein byreference in its entirety without disclaimer and for all purposes.

STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSOREDRESEARCH AND DEVELOPMENT

NOT APPLICABLE

REFERENCE TO A “SEQUENCE LISTING,” A TABLE, OR A COMPUTER PROGRAMLISTING APPENDIX SUBMITTED ON A COMPACT DISK

NOT APPLICABLE

1. BACKGROUND OF THE INVENTION

1.1 Field of the Invention

The present invention relates generally to the fields of cancerdiagnosis and therapy. More particularly, it concerns the surprisingdiscovery of compositions and methods for the detection andimmunotherapy of hematological malignancies, and particularly, B cellleukemias, and lymphomas and multiple myelomas. The invention providesnew, effective methods, compositions and kits for eliciting immune andT-cell response to antigenic polypeptides, and antigenic peptidefragments isolated therefrom, and methods for the use of suchcompositions for diagnosis, detection, treatment, monitoring, and/orprevention of various types of human hematological malignancies. Inparticular, the invention provides polypeptide, peptide, antibody,antigen binding fragment, hybridoma, host cell, vector, andpolynucleotide compounds and compositions for use in identification anddiscrimination between various types of hematological malignancies, andmethods for the detection, diagnosis, prognosis, monitoring, and therapyof such conditions in an affected animal.

1.2 Description of Related Art

1.2.1 Hematological Malignancies

Hematological malignancies, such as leukemias and lymphomas, areconditions characterized by abnormal growth and maturation ofhematopoietic cells. Leukemias are generally neoplastic disorders ofhematopoietic stem cells, and include adult and pediatric acute myeloidleukemia (AML), chronic myeloid leukemia (CML), acute lymphocyticleukemia (ALL), chronic lymphocytic leukemia (CLL) and secondaryleukemia. Among lymphomas, there are two distinct groups: non-Hodgkin'slymphoma (NHL) and Hodgkin's disease. NHLs are the result of a clonalexpansion of B- or T-cells, but the molecular pathogenesis of Hodgkin'sdisease, including lineage derivation and clonality, remains obscure.Other hematological malignancies include myelodysplastic syndromes(MDS), myeloproliferative syndromes (MPS) and myeloma. Hematologicalmalignancies are generally serious disorders, resulting in a variety ofsymptoms, including bone marrow failure and organ failure.

NHLs are the sixth most common cause of cancer related deaths in theUnited States. Only prostate, breast, lung, colorectal and bladdercancer currently exceed lymphoma in annual incidence. In 1995, more than45,000 new NHLs were diagnosed, and over 21,000 patients died of thesediseases. The average age of lymphoma patients is relatively young (42years), and the resulting number of years of life lost to these diseasesrenders NHLs fourth in economic impact among cancers in the UnitedStates. In the past 15 years, the American Cancer Society reported a 50%increase in the incidence of NHLs, one of the largest increases for anycancer group. Much of this increase has been attributed to thedevelopment of lymphomas in younger men who have acquired AIDS.Lymphomas are also the third most common childhood malignancy andaccount for approximately 10% of cancers in children. The survival rate(all ages) varies from 73% (low risk) to 26% (high risk).

1.3 Deficiencies in the Prior Art

Treatment for many hematological malignancies, including leukemias andlymphomas, remains difficult, and existing therapies are not universallyeffective. While treatments involving specific immunotherapy appear tohave considerable potential, such treatments have been limited by thesmall number of known malignancy-associated antigens. Moreover theability to detect such hematological malignancies in their early stagescan be quite difficult depending upon the particular malady. The lack ofa sufficient number of specific diagnostic and prognostic markers of thediseases, and identification of cells and tissues that can be affected,has significantly limited the field of oncology.

Accordingly, there remains a need in the art for improved methods fordetecting, screening, diagnosis and treatment of hematologicalmalignancies such as B cell leukemias and lymphomas and multiplemyelomas. The present invention fulfills these and other inherent needsin the field, and provides significant advantages in the detection ofcells, and cell types that express one or more polypeptides that havebeen shown to be over-expressed in one or more of such hematologicalmalignancies.

2. SUMMARY OF THE INVENTION

The present invention addresses the foregoing long-felt need and otherdeficiencies in the art by identifying new and effective strategies forthe identification, detection, screening, diagnosis, prognosis,prophylaxis, therapy, and immunomodulation of one or more hematologicalmalignancies, and in particular, B cell leukemias and lymphomas, andmultiple myelomas.

The present invention is based, in part, upon the surprising andunexpected discovery that certain previously unknown or unidentifiedhuman polypeptides, peptides, and antigenic fragments derived therefromhave now been identified that are overexpressed in one or more types ofhematological malignancies. The genes encoding several of thesepolypeptides are now identified and obtained in isolated form, and havebeen characterized using a series of molecular biology methodologiesincluding subtractive library analysis, microarray screening,polynucleotide sequencing, peptide and epitopic identification andcharacterization, as well as expression profiling, and in vitro wholegene cell priming. A set of these polynucleotides, and the polypeptides,peptides, and antigenic fragments they encode are now identified andimplicated in the complex processes of hematological malignancy diseaseonset, progression, and/or outcome, and in particular, diseases such asleukemias and lymphomas.

The inventors have further demonstrated that a number of thesepolynucleotides, and their encoded polypeptides, as well as antibodies,antigen presenting cells, T cells, and the antigen binding fragmentsderived from such antibodies are useful in the development ofparticularly advantageous compositions and methods for the detection,diagnosis, prognosis, prophylaxis and/or therapy of one or more of thesediseases, and particularly those conditions that are characterized by(a) increased, altered, elevated, or sustained expression of one or morepolynucleotides that comprise at least a first sequence region thatcomprises a nucleic acid sequence as disclosed in any one of SEQ IDNOs:11,000-11,038 and 11,291-11,292 or (b) an increased, altered,elevated, or sustained biological activity of one or more polypeptidesthat comprise at least a first sequence region that comprises an aminoacid sequence as encoded by any one of the above polynucleotides ordisclosed in any one of SEQ ID NOs:11,039-11,058 and 11293.

The present invention also provides methods and uses for one or more ofthe disclosed peptide, polypeptide, antibody, antigen binding fragment,and polynucleotide compositions of the present invention in generatingan immune response or in generating a T-cell response in an animal, andin particular in a mammal such as a human. The invention also providesmethods and uses for one or more of these compositions in theidentification, detection, and quantitation of hematological malignancycompositions in clinical samples, isolated cells, whole tissues, andeven affected individuals. The compositions and methods disclosed hereinalso may be used in the preparation of one or more diagnostic reagents,assays, medicaments, or therapeutics, for diagnosis and/or therapy ofsuch diseases.

In a first important embodiment, there is provided a compositioncomprising at least a first isolated peptide or polypeptide thatcomprises an amino acid sequence that is at least about 80%, about 81%,about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%,about 95%, about 96%, about 97%, about 98%, or about 99% identical tothe amino acid sequence encoded by any one of the above polynucleotidesor disclosed in any one of SEQ ID NOs:11,039-11,058 and 11,293.Exemplary preferred sequences are those that comprise at least a firstcoding region that comprises an amino acid sequence that is at leastabout 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about91%, about 92%, about 93%, or about 94% identical to the amino acidsequence as encoded by any one of the above polynucleotides or disclosedin any one of SEQ ID NOs:11,039-11,058 and 11,293, with those sequencesthat comprise at least a first coding region that comprises an aminoacid sequence that is at least about 95%, about 96%, about 97%, about98%, or about 99% identical to the amino acid sequence as encoded by anyone of the above polynucleotides or disclosed in any one of SEQ IDNOs:11,039-11,058, and 11,293 being examples of particularly preferredsequences in the practice of the present invention. Likewise, peptideand polypeptide compounds and compositions are also provided thatcomprise, consist essentially of, or consist of the amino acid sequenceas encoded by any one of the above polynucleotides or disclosed in anyone of SEQ ID NOs:11,039-11,058, and 11,293.

In a similar fashion, there are also embodiments disclosed herein thatprovide compositions and methods for the detection, diagnosis,prognosis, prophylaxis, treatment, and therapy of B cell leukemia,lymphoma and multiple myeloma. Exemplary preferred peptide andpolypeptide compounds and compositions relating to this aspect of theinvention include, but are not limited to, those peptide and polypeptidecompounds or compositions that comprise at least a first isolatedpeptide or polypeptide that comprises an amino acid sequence that is atleast about 80%, about 81%, about 82%, about 83%, about 84%, about 85%,about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%,or about 99% identical to the amino acid sequence as encoded by any oneof the above polynucleotides or disclosed in any one of SEQ IDNOs:11,039-11,058, and 11,293 and those that comprise at least a firstcoding region that comprises an amino acid sequence that is at leastabout 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about91%, about 92%, about 93%, or about 94% identical to the amino acidsequence as encoded by any one of the above polynucleotides or disclosedin any one of SEQ ID NOs:11,039-11,058 and 11,293, and even thosesequences that comprise at least a first coding region that comprises anamino acid sequence that is at least about 95%, about 96%, about 97%,about 98%, or about 99% identical to the amino acid sequence as encodedby any one of the above polynucleotides or disclosed in any one of SEQID NOs:11,039-11,058, and 11,293.

Exemplary peptides of the present invention may be of any suitablelength, depending upon the particular application thereof, and encompassthose peptides that are about 9, about 10, about 15, about 20, about 25,about 30, about 35, about 40, about 45, about 50, about 55, about 60,about 65, about 70, about 75, about 80, about 85, about 90, about 95, orabout 100 or so amino acids in length. Of course, the peptides of theinvention may also encompass any intermediate lengths or integers withinthe stated ranges.

Exemplary polypeptides and proteins of the present invention may be ofany suitable length, depending upon the particular application thereof,and encompass those polypeptides and proteins that are about 100, about150, about 200, about 250, about 300, about 350, or about 400 or soamino acids in length. Of course, the polypeptides and proteins of theinvention may also encompass any intermediate lengths or integers withinthe stated ranges.

The peptides, polypeptides, proteins, antibodies, and antigen bindingfragments of the present invention will preferably comprise a sequenceof at least about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70,75, 80, 85, 90, 95 or 100 contiguous amino acids from any one of thepeptides as encoded by any one of the above polynucleotides or disclosedin any one of SEQ ID NOs:11,039-11,058 and 11,293.

Furthermore, the polypeptides, proteins, antibodies, and antigen bindingfragments of the present invention will even more preferably comprise atleast a first isolated coding region that comprises a sequence of atleast about 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, or 200contiguous amino acids from any one of the peptides as encoded by anyone of the above polynucleotides or disclosed in any one of SEQ IDNOs:11,039-11,058, and 11,293.

Likewise, the polypeptides, proteins, antibodies, and antigen bindingfragments of the present invention may comprise at least a firstisolated coding region that comprises a substantially longer sequence,such as for example, one of at least about 200, 220, 240, 260, 280, or300 or more contiguous amino acids from any one of the peptides asencoded by any one of the above polynucleotides or disclosed in any oneof SEQ ID NOs:11,039-11,058, and 11,293.

In illustrative embodiments, and particularly in those embodimentsconcerning methods and compositions relating to B cell leukemias,lymphomas and multiple myelomas, the polypeptides of the inventioncomprise an amino acid sequence that (a) comprises, (b) consistsessentially of, or (c) consists of, the amino acid sequence encoded byany one of the above polynucleotides or disclosed in any one of SEQ IDNOs:11,039-11,058, and 11,293.

The polypeptides and proteins of the invention preferably comprise anamino acid sequence that is encoded by at least a first nucleic acidsegment that comprises an at least 21, 22, 23, 24, 25, 26, 27, 28, 29,or 30 contiguous nucleotide sequence of any one of SEQ IDNOs:11,000-11,038 and 11,291-11,292.

The polypeptides and proteins of the invention may also preferablycomprise an amino acid sequence encoded by at least a first nucleic acidsegment that comprises an at least about 31, 32, 33, 34, 35, 36, 37, 38,39, or 40 contiguous nucleotide sequence of any one of SEQ IDNOs:11,000-11,038, and 11,292-11,292. The polypeptides and proteins ofthe invention may also preferably comprise one or more coding regionsthat comprise an amino acid sequence encoded by at least a first nucleicacid segment that comprises an at least about 41, 42, 43, 44, 45, 46,47, 48, 49, or 50 contiguous nucleotide sequence of any one of SEQ IDNOs:11,000-11,038, and 11,291-11,292. The polypeptides and proteins ofthe invention may also preferably comprise one or more coding regionsthat comprise an amino acid sequence encoded by at least a first nucleicacid segment that comprises an at least about 51, 52, 53, 54, 55, 56,57, 58, 59, or 60 contiguous nucleotide sequence of any one of SEQ IDNOs:11,000-11,038, ans 11,291-11,292. The polypeptides and proteins ofthe invention may also preferably comprise one or more coding regionsthat comprise an amino acid sequence encoded by at least a first nucleicacid segment that comprises an at least about 70, 80, 90, 100, 110, 120,130, 140 or 150 contiguous nucleotide sequence of any one of SEQ IDNOs:11,000-11,038, and 11,291-11,292. The polypeptides and proteins ofthe invention may also preferably comprise one or more coding regionsthat comprise an amino acid sequence encoded by at least a first nucleicacid segment that comprises an at least about 175, 200, 225, 250, 275,300, 325, 350, 375, or 400 contiguous nucleotide sequence of any one ofSEQ ID NOs:11,000-11,038, and 11,291-11,292. The polypeptides andproteins of the invention may also preferably comprise one or morecoding regions that comprise an amino acid sequence encoded by at leasta first nucleic acid segment that comprises an at least about 500, 600,700, 800, 900, 1000, 1100, 1200, 1300, 1400, or 1500 contiguousnucleotide sequence of anyone of SEQ ID NOs:11,000-11,038, and11,291-11,292.

In a second important embodiment, there is provided a compositioncomprising at least a first isolated polynucleotide that comprises anucleic acid sequence that is at least about 80%, about 81%, about 82%,about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%,about 96%, about 97%, about 98%, or about 99% identical to the nucleicacid sequence of any one of SEQ ID NOs:11,000-11,038, and 11,291-11,292.Exemplary preferred sequences are those that comprise a nucleic acidsequence that is at least about 85%, about 86%, about 87%, about 88%,about 89%, about 90%, about 91%, about 92%, about 93%, or about 94%identical to the nucleic acid sequence of any one of SEQ IDNOs:11,000-11,038 and 11,291-11,292, with those sequences that compriseat least a nucleic acid sequence that is at least about 95%, about 96%,about 97%, about 98%, or about 99% identical to the nucleic acidsequence of any one of SEQ ID NOs:11,000-11,038, and 11,291-11,292 beingexamples of particularly preferred sequences in the practice of thepresent invention.

In embodiments that relate particularly to compositions and methods forthe detection, diagnosis, prognosis, prophylaxis, treatment, and therapyof B cell leukemias, lymphomas, and multiple myelomas exemplarypreferred polynucleotide compositions include those compositions thatcomprise at least a first isolated nucleic acid segment that comprises asequence that is at least about 80%, about 81%, about 82%, about 83%,about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%,about 97%, about 98%, or about 99% identical to the nucleic acidsequence of any one of SEQ ID NOs:11,000-11,038, and 11,291-11,292. Suchpolynucleotides will preferably comprise one or more isolated codingregion, each of which may (a) comprise, (b) consist essentially of, or(c) consist of, the nucleic acid sequence of SEQ ID NOs:11,000-11,038,and 11,291-11,292.

Exemplary polynucleotides of the present invention may be of anysuitable length, depending upon the particular application thereof, andencompass those polynucleotides that (a) are at least about, or (b)comprise at least a first isolated nucleic acid segment that is at leastabout 27, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 120, 140, 150, 160,170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300,320, 340, 360, 380, 400, 420, 440, 460, 480, 500, 520, 540, 560, 580,600, 625, 650, 675, 700, 750, 800, 850, 900, 950, or 1000 or so nucleicacids in length, as well as longer polynucleotides that (a) are at leastabout, or (b) comprise at least a first isolated nucleic acid segmentthat is at least about 1000, 1025, 1050, 1075, 1100, 150, 1200, 1250,1300, 1350, 1400, 1450, 1500, 1550, 1600, 1650, 1700, 1750, 1800, 1850,1900, 1950, 2000, 2100, 2200, 2300, 2400, 2500, 2600, 2700, 2800, 2900,or 3000 or so nucleic acids in length, as well as substantially largerpolynucleotides. Of course, the polynucleotides and nucleic acidsegments of the invention may also encompass any intermediate lengths orintegers within the stated ranges.

The compositions of the present invention may comprise a singlepolypeptide or polynucleotide, or alternatively, may comprise two ormore such hematological malignancy compounds, such as for example, twoor more polypeptides, two or more polynucleotides, or even combinationsof one or more peptides or polypeptides, along with one or morepolynucleotides. When two or more polypeptides are contemplated forparticular applications, the second and/or third and/or fourth, etc.isolated peptides and/or polypeptides will preferably comprise an aminoacid sequence that is at least about 91%, 93%, 95%, 97%, or 99%identical to the amino acid sequence encoded by any one of the abovepolynucleotides or disclosed in any one of SEQ ID NOs:11,039-11,058, and11,293. Alternatively, the polynucleotides of the invention may compriseone or more coding regions that encode a first fusion protein orpeptide, such as an adjuvant-coding region fused in correct readingframe to one or more of the disclosed hematological malignancy peptidesor polypeptides. Alternatively, the fusion protein may comprise ahematological malignancy polypeptide or peptide fused, in correctreading frame, to a detectable protein or peptide, or to animmunostimulant protein or peptide, or other such construct. Fusionproteins such as these are particularly useful in those embodimentsrelating to diagnosis, detection, and therapy of one or more of thehematological malignancies as discussed herein.

The invention also provides a composition comprising at least a firsthybridoma cell line that produces a monoclonal antibody havingimmunospecificity for one or more of the peptides or polypeptides asdisclosed herein, or at least a first monoclonal antibody, or anantigen-binding fragment thereof, that has immunospecificity for such apeptide or polypeptide. The antigen binding fragments may comprise alight chain variable region, a heavy-chain variable region, a Fabfragment, a F(ab)₂ fragment, an Fv fragment, an scFv fragment, or anantigen-binding fragment of such an antibody.

The invention also provides a composition comprising at least a firstisolated antigen-presenting cell that expresses a peptide or polypeptideas disclosed herein, or a plurality of isolated T cells thatspecifically react with such a peptide or polypeptide. Such pluralitiesof isolated T cells may be stimulated or expanded by contacting the Tcells with one or more peptides or polypeptides as described herein. TheT cells may be cloned prior to expansion, and may be obtained from bonemarrow, a bone marrow fraction, peripheral blood, or a peripheral bloodfraction from a healthy mammal, or from a mammal that is afflicted withat least a first hematological malignancy such as leukemia or lymphoma.

As described above, the isolated polypeptides of the invention may be onthe order of from 9 to about 1000 amino acids in length, oralternatively, may be on the order of from 50 to about 900 amino acidsin length, from 75 to about 800 amino acids in length, from 100 to about700 amino acids in length, or from 125 to about 600 amino acids inlength, or any other such suitable range.

The isolated nucleic acid segments that encode such isolatedpolypeptides may be on the order of from 27 to about 10,000 nucleotidesin length, from 150 to about 8000 nucleotides in length, from 250 toabout 6000 nucleotides in length, from 350 to about 4000 nucleotides inlength, or from 450 to about 2000 nucleotides in length, or any othersuch suitable range.

The nucleic acid segment may be operably positioned under the control ofat least a first heterologous, recombinant promoter, such as atissue-specific, cell-specific, inducible, or otherwise regulatedpromoter. Such promoters may be further controlled or regulated by thepresence of one or more additional enhancers or regulatory regionsdepending upon the particular cell type in which expression of thepolynucleotide is desired. The polynucleotides and nucleic acid segmentsof the invention may also be comprised within a vector, such as aplasmid, or viral vector. The polypeptides and polynucleotides of theinvention may also be comprised within a host cell, such as arecombinant host cell or a human host cell such as a blood or bonemarrow cell.

The polynucleotides of the invention may comprise at least a firstisolated nucleic acid segment operably attached, in frame, to at least asecond isolated nucleic acid segment, such that the polynucleotideencodes a fusion protein in which the first peptide or polypeptide islinked to the second peptide or polypeptide.

The polypeptides of the present invention may comprise a contiguousamino acid of any suitable length, such as for example, those of about2000, about 1900, about 1850, about 1800, about 1750, about 1700, about1650, about 1600, about 1550, about 1500, about 1450, about 1400, about1350, about 1300, about 1250, about 1200, about 1150, about 1100 aminoacids, or about 1000 or so amino acids in length. Likewise, thepolypeptides and peptides of the present invention may comprise slightlyshorter contiguous amino acid coding regions, such as for example, thoseof about 950, about 900, about 850, about 800, about 750, about 700,about 650, about 600, about 550, about 500, about 450, about 400, about350, about 300, about 250, about 200, about 150, or even about 100 aminoacids or so in length.

In similar fashion, the polypeptides and peptides of the presentinvention may comprise even smaller contiguous amino acid codingregions, such as for example, those of about 95, about 90, about 85,about 80, about 75, about 70, about 65, about 60, about 55, about 50,about 45, about 40, about 35, about 30, about 25, about 20, about 15, oreven about 9 amino acids or so in length.

In all such embodiments, those peptides and polypeptides havingintermediate lengths including all integers within the preferred ranges(e.g., those peptides and polypeptides that comprise at least a firstcoding region of at least about 94, about 93, about 92, about 91, about89, about 88, about 87, about 86, about 84, about 83, about 82, about81, about 79, about 78, about 77, about 76, about 74, about 73, about72, about 71, about 69, about 68, about 67, about 66 or so amino acidsin length, etc.) are all contemplated to fall within the scope of thepresent invention.

In particular embodiments, the peptides and polypeptides of the presentinvention may comprise a sequence of at least about 9, or about 10, orabout 11, or about 12, or about 13, or about 14, or about 15, or about16, or about 17, or about 18, or about 19, or about 20, or about 21, orabout 22, or about 23, or about 24, or about 25, or about 26, or about27, or about 28, or about 29, or about 30, or about 31, or about 32, orabout 33, or about 34, or about 35, or about 36, or about 37, or about38, or about 39, or about 40, or about 41, or about 42, or about 43, orabout 44, or about 45, or about 46, or about 47, or about 48, or about49, or about 50 contiguous amino acids as disclosed in any one or moreof the peptides encoded by any one of the above polynucleotides ordisclosed in any one of SEQ ID NOs:11,039-11,058, and 11,293 herein.

In other embodiments, the peptides and polypeptides of the presentinvention may comprise a sequence of at least about 51, or about 52, orabout 53, or about 54, or about 55, or about 56, or about 57, or about58, or about 59, or about 60, or about 61, or about 62, or about 63, orabout 64, or about 65, or about 66, or about 67, or about 68, or about69, or about 70, or about 71, or about 72, or about 73, or about 74, orabout 75, or about 76, or about 77, or about 78, or about 79, or about80, or about 81, or about 82, or about 83, or about 84, or about 85, orabout 86, or about 87, or about 88, or about 89, or about 90, about 91,or about 92, or about 93, or about 94, or about 95, or about 96, orabout 97, or about 98, or about 99, or 100 contiguous amino acids asdisclosed in any one or more of the peptides encoded by any one of theabove polynucleotides or disclosed in any one of SEQ IDNOs:11,039-11,058, and 11,293 herein.

In still other embodiments, the preferred peptides and polypeptides ofthe present invention comprise a sequence of at least about 100, 125,150, 175, 200, 225, 250, 275, 300, 325, 350, 375, or 400 or morecontiguous amino acids as disclosed in any one or more of the peptidesencoded by any one of the above polynucleotides or disclosed in any oneof SEQ ID NOs:11,039-11,058, and 11,293 herein.

The polypeptides of the invention typically will comprise at least afirst contiguous amino acid sequence according to any one of thepeptides encoded by any one of the above polynucleotides or disclosed inany one of SEQ ID NOs:11,039-11,058, and 11,293, but may also,optionally comprise at least a second, at least a third, or even atleast a fourth or greater contiguous amino acid sequence according toany one of the peptides encoded by any one of the above polynucleotidesor disclosed in any one of SEQ ID NOs:11,039-11,058, and 11,293. Asingle polypeptide may contain only a single coding region, oralternatively, a single polypeptide may comprise a plurality ofidentical or distinctly different contiguous amino acid sequences inaccordance with any one of the peptides encoded by any one of the abovepolynucleotides or disclosed in any one of SEQ ID NOs:11,039-11,058, and11,293. In fact, the polypeptide may comprise a plurality of the samecontiguous amino acid sequences, or they may comprise one or moredifferent contiguous amino acid sequences of any of the peptides encodedby any one of the above polynucleotides or disclosed in any one of SEQID NOs:11,039-11,058, and 11,293. For example, a single polypeptide cancomprise a single contiguous amino acid sequence from one or more of thepeptides encoded by any one of the above polynucleotides or disclosed inany one of SEQ ID NOs:11,039-11,058, and 11,293, or alternatively, maycomprise two or more distinctly different contiguous amino acidsequences from one or more of the peptides encoded by any one of theabove polynucleotides or disclosed in any one of SEQ IDNOs:11,039-11,058, and 11,293. In fact, the polypeptide may comprise 2,3, 4, or even 5 distinct contiguous amino sequences of any one of thepeptides encoded by any one of the above polynucleotides or disclosed inany one of SEQ ID NOs: 11,039-11,058 and 11,293. Alternatively, a singlepolypeptide may comprise 2, 3, 4, or even 5 distinct coding regions. Forexample, a polypeptide may comprise at least a first coding region thatcomprises a first contiguous amino acid sequence of any one of thepeptides encoded by any one of the above polynucleotides or disclosed inany one of SEQ ID NOs:11,039-11,058, and 11,293, and at least a secondcoding region that comprises a second contiguous amino acid sequence ofany one of the peptides encoded by any one of the above polynucleotidesor disclosed in any one of SEQ ID NOs:11,039-11,058, and 11,293. Incontrast, a polypeptide may comprise at least a first coding region thatcomprises a first contiguous amino acid sequence of any one of thepeptides encoded by any one of the above polynucleotides or disclosed inany one of SEQ ID NOs:11,039-11,058, and 11,293, and at least a secondcoding region that comprises a second distinctly different peptide orpolypeptide, such as for example, an adjuvant or an immunostimulantpeptide or polypeptide.

In such cases, the two coding regions may be separate on the samepolypeptide, or the two coding regions may be operatively attached, eachin the correct reading frame, such that a fusion polypeptide isproduced, in which the first amino acid sequence of the first codingregion is linked to the second amino acid sequence of the second codingregion.

Throughout this disclosure, a phrase such as “a sequence as disclosed inSEQ ID NO:11,000 to SEQ ID NO:11,004” is intended to encompass any andall contiguous sequences disclosed by any one of these sequenceidentifiers. That is to say, “a sequence as disclosed in any of SEQ IDNO:11,000 through SEQ ID NO:11,004” means any sequence that is disclosedin any one of SEQ BD NO:11,000, SEQ ID NO:11,001, SEQ ID NO:11,002, SEQID NO:11,003, or SEQ ID NO:11,004. Likewise, “a sequence as disclosed inany of SEQ ID NOs:25 to 37” means any sequence that is disclosed in anyone of SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ IDNO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ IDNO:34, SEQ ID NO:35, SEQ ID NO:36, or SEQ ID NO:37, and so forth.

Likewise, a phrase such as “at least a first sequence from any one ofSEQ ID NO:55 to SEQ ID NO:62” is intended to refer to a first sequencethat is disclosed in any one of SEQ ID NO:55, SEQ ID NO:56, SEQ IDNO:57, SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:61, or SEQ IDNO:62.

It will also be understood that the kits, and compositions of thepresent invention comprise in an overall and general sense at least oneor more particular polynucleotides, polypeptides, and peptides thatcomprise one or more contiguous sequence regions from one or more of thenucleic acid sequences disclosed herein in SEQ ID NOs:11,000-11,038, and11,291-11,292 or from one or more of the amino acid sequences encoded byany one of the above polynucleotides or disclosed in any one of SEQ IDNOs:11,039-11,058, and 11,293, and that such peptide, polypeptide andpolynucleotide compositions may be used in one or more of the particularmethods and uses disclosed herein for the diagnosis, detection,prophylaxis, and therapy of one or more hematological cancers, and inparticular, lymphomas of a variety of specific types. It will also beunderstood to the skilled artisan having benefit of the teachings of thepresent Specification, that the peptide and polypeptide compositions maybe used to generate a T cell or an immune response in an animal, andthat such compositions may also be administered to an animal from whichimmunospecific antibodies and antigen binding fragments may be isolatedor identified that specifically bind to such peptides or polypeptides.Such an artisan will also recognize that the polynucleotides identifiedby the present disclosure may be used to produce such peptides,polypeptides, antibodies, and antigen binding fragments, by recombinantprotein production methodologies that are also within the capability ofthe skilled artisan having benefit of the specific amino acid andnucleic acid sequences provided herein.

Likewise, it will be understood by a skilled artisan in the field, thatone or more of the disclosed compositions may used in one or morediagnostic or detection methodologies to identify certain antibodies,peptides, polynucleotides, or polypeptides in a biological sample, in ahost cell, or even within the body or tissues of an animal. It will beunderstood by a skilled artisan in the field, that one or more of thedisclosed nucleic acid or amino acid compositions may used in thepreparation or manufacture of one or more medicaments for use in thediagnosis, detection, prognosis, prophylaxis, or therapy of one or morehematological malignancies in an animal, and particularly thosemalignant conditions disclosed and claimed herein.

It will also be readily apparent to those of skill in the art, that themethods, kits, and uses, of the present invention preferably employ oneor more of the compounds and/or compositions disclosed herein thatcomprise one or more contiguous nucleotide sequences as may be presentedin SEQ ID NOs:11,000-11,038, and 11,291-11,292, of the attached sequencelisting.

Likewise, it will also be readily apparent to those of skill in the art,that the methods, kits, and uses, of the present invention may alsoemploy one or more of the compounds and compositions disclosed hereinthat comprise one or more contiguous amino acid sequences of any of thepeptides encoded by any one of the above polynucleotides or presented inany one of SEQ ID NOs:11,039-11,058 of the attached sequence listing.

3. BRIEF DESCRIPTION OF THE DRAWINGS AND THE APPENDICES

The invention may be understood by reference to the followingdescription taken in conjunction with the accompanying drawings, inwhich like reference numerals identify like elements, and in which:

FIG. 1 illustrates a schematic outline of the microarray chip technologyapproach used to identify the cDNA targets of the present invention asdescribed Section 5.1.

FIG. 2 illustrates a schematic outline of the general protocol for invitro whole gene CD8⁺ T cell priming procedure used to generateantigen-specific lines and to identify clones of interest.

FIG. 3 illustrates a schematic outline of the general protocol for invitro whole gene CD4⁺ T cell priming procedure used to generateantigen-specific lines and to identify clones of interest.

FIG. 4 illustrates the panel of probes used to identify cDNAs that areoverexpressed in lymphoma cells.

FIG. 5 illustrates the results of analyzing SEQ ID NO:9611 with theprogram TSITES.

FIG. 6 illustrates the presence of Ly1448P-specific serum antibodies inlymphoma patients.

FIG. 7 diagrammatically illustrates the 18 LY1448P exons present onchromosome 1 and the location of these exons in several splice variants.

FIG. 8 depicts the Ly1448P splice forms and the exons used in each.

FIG. 9 diagrammatically depicts the structure of each of the Ly1448Psplice forms.

4. DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

In order that the invention herein described may be more fullyunderstood, the following description of various illustrativeembodiments is set forth.

The present invention is generally directed to compositions and methodsfor the immunotherapy and diagnosis of Hematological malignancies, suchas B cell leukemias and lymphomas and multiple myelomas.

4.1 Methods of Nucleic Acid Delivery and DNA Transfection

In certain embodiments, it is contemplated that one or more RNA or DNAand/or substituted polynucleotide compositions disclosed herein will beused to transfect an appropriate host cell. Technology for introductionof RNAs and DNAs, and vectors comprising them into suitable host cellsis well known to those of skill in the art. In particular, suchpolynucleotides may be used to genetically transform one or more hostcells, when therapeutic administration of one or more active peptides,compounds or vaccines is achieved through the expression of one or morepolynucleotide constructs that encode one or more therapeutic compoundsof interest.

A variety of means for introducing polynucleotides and/or polypeptidesinto suitable target cells is known to those of skill in the art. Forexample, when polynucleotides are contemplated for delivery to cells,several non-viral methods for the transfer of expression constructs intocultured mammalian cells are available to the skilled artisan for hisuse. These include, for example, calcium phosphate precipitation (Grahamand Van Der Eb, 1973; Chen and Okayama, 1987; Rippe et al., 1990);DEAE-dextran precipitation (Gopal, 1985); electroporation (Wong andNeumann, 1982; Fromm et al., 1985; Tur-Kaspa et al, 1986; Potter et al.,1984; Suzuki et al., 1998; Vanbever et al., 1998), direct microinjection(Capecchi, 1980; Harland and Weintraub, 1985), DNA-loaded liposomes(Nicolau and Sene, 1982; Fraley et al., 1979; Takakura, 1998) andlipofectamine-DNA complexes, cell sonication (Fechheimer et al., 1987),gene bombardment using high velocity microprojectiles (Yang et al.,1990; Klein et al., 1992), and receptor-mediated transfection (Curiel etal., 1991; Wagner et al., 1992; Wu and Wu, 1987; Wu and Wu, 1988). Someof these techniques may be successfully adapted for in vivo or ex vivouse.

A bacterial cell, a yeast cell, or an animal cell transformed with oneor more of the disclosed expression vectors represent an importantaspect of the present invention. Such transformed host cells are oftendesirable for use in the expression of the various DNA gene constructsdisclosed herein. In some aspects of the invention, it is oftendesirable to modulate, regulate, or otherwise control the expression ofthe gene segments disclosed herein. Such methods are routine to those ofskill in the molecular genetic arts. Typically, when increased orover-expression of a particular gene is desired, various manipulationsmay be employed for enhancing the expression of the messenger RNA,particularly by using an active promoter, and in particular, atissue-specific promoter such as those disclosed herein, as well as byemploying sequences, which enhance the stability of the messenger RNA inthe particular transformed host cell.

Typically, the initiation and translational termination region willinvolve stop codon(s), a terminator region, and optionally, apolyadenylation signal. In the direction of transcription, namely in the5′ to 3′ direction of the coding or sense sequence, the construct willinvolve the transcriptional regulatory region, if any, and the promoter,where the regulatory region may be either 5′ or 3′ of the promoter, theribosomal binding site, the initiation codon, the structural gene havingan open reading frame in phase with the initiation codon, the stopcodon(s), the polyadenylation signal sequence, if any, and theterminator region. This sequence as a double strand may be used byitself for transformation of a microorganism or eukaryotic host, butwill usually be included with a DNA sequence involving a marker, wherethe second DNA sequence may be joined to the expression construct duringintroduction of the DNA into the host.

Where no functional replication system is present, the construct willalso preferably include a sequence of at least about 30 or about 40 orabout 50 base pairs (bp) or so, preferably at least about 60, about 70,about 80, or about 90 to about 100 or so bp, and usually not more thanabout 500 to about 1000 or so bp of a sequence homologous with asequence in the host. In this way, the probability of legitimaterecombination is enhanced, so that the gene will be integrated into thehost and stably maintained by the host. Desirably, the regulatoryregions of the expression construct will be in close proximity to (andalso operably positioned relative to) the selected therapeutic geneproviding for complementation as well as the gene providing for thecompetitive advantage. Therefore, in the event that the therapeutic geneis lost, the resulting organism will be likely to also lose the geneproviding for the competitive advantage, so that it will be unable tocompete in the environment with the gene retaining the intact construct.

The selected therapeutic gene can be introduced between thetranscriptional and translational initiation region and thetranscriptional and translational termination region, so as to be underthe regulatory control of the initiation region. This construct may beincluded in a plasmid, which will include at least one replicationsystem, but may include more than one, where one replication system isemployed for cloning during the development of the plasmid and thesecond replication system is necessary for functioning in the ultimatehost, in this case, a mammalian host cell. In addition, one or moremarkers may be present, which have been described previously. Whereintegration is desired, the plasmid will desirably include a sequencehomologous with the host genome.

Genes or other nucleic acid segments, as disclosed herein, can beinserted into host cells using a variety of techniques that are wellknown in the art. Five general methods for delivering a nucleic segmentinto cells have been described: (1) chemical methods (Graham and Van DerEb, 1973); (2) physical methods such as microinjection (Capecchi, 1980),electroporation (U.S. Pat. No. 5,472,869; Wong and Neumann, 1982; Frommet al., 1985), microprojectiles bombardment (U.S. Pat. No. 5,874,265,specifically incorporated herein by reference in its entirety), “genegun” (Yang et al., 1990); (3) viral vectors (Eglitis and Anderson,1988); (4) receptor-mediated mechanisms (Curiel et al., 1991; Wagner etal., 1992); and (5) bacterial-mediated transformation

4.2 Hematological Malignancy Related-Specific Antibodies andAntigen-Binding Fragments Thereof

The present invention further provides antibodies and antigen-bindingfragments thereof, that specifically bind to (or are immunospecific for)at least a first peptide or peptide variant as disclosed herein. As usedherein, an antibody or an antigen-binding fragment is said to“specifically bind” to a peptide if it reacts at a detectable level(within, for example, an ELISA) with the peptide, and does not reactdetectably with unrelated peptides or proteins under similar conditions.As used herein, “binding” refers to a non-covalent association betweentwo separate molecules such that a “complex” is formed. The ability tobind may be evaluated by, for example, determining a binding constantfor the formation of the complex. The binding constant is the valueobtained when the concentration of the complex is divided by the productof the component concentrations. In the context of the presentinvention, in general, two compounds are said to “bind” when the bindingconstant for complex formation exceeds about 10³ L/mol. The bindingconstant maybe determined using methods well known in the art.

Any agent that satisfies the above requirements may be a binding agent.In illustrative embodiments, a binding agent is an antibody or anantigen-binding fragment thereof. Such antibodies may be prepared by anyof a variety of techniques known to those of ordinary skill in the art(Harlow and Lane, 1988). In general, antibodies can be produced by cellculture techniques, including the generation of monoclonal antibodies asdescribed herein, or via transfection of antibody genes into suitablebacterial or mammalian cell hosts, in order to allow for the productionof recombinant antibodies. In one technique, an immunogen comprising thepeptide is initially injected into any of a wide variety of mammals(e.g., mice, rats, rabbits, sheep or goats). In this step, the peptidesof this invention may serve as the immunogen without modification.Alternatively, particularly for relatively short peptides, a superiorimmune response may be elicited if the peptide is joined to a carrierprotein, such as bovine serum albumin or keyhole limpet hemocyanin. Theimmunogen is injected into the animal host, preferably according to apredetermined schedule incorporating one or more booster immunizations,and the animals are bled periodically. Polyclonal antibodies specificfor the peptide may then be purified from such antisera by, for example,affinity chromatography using the peptide coupled to a suitable solidsupport.

Monoclonal antibodies specific for the antigenic peptide of interest maybe prepared, for example, using the technique of Kohler and Milstein(1976) and improvements thereto. Briefly, these methods involve thepreparation of immortal cell lines capable of producing antibodieshaving the desired specificity (i.e., reactivity with the peptide ofinterest). Such cell lines may be produced, for example, from spleencells obtained from an animal immunized as described above. The spleencells are then immortalized by, for example, fusion with a myeloma cellfusion partner, preferably one that is syngeneic with the immunizedanimal. A variety of fusion techniques may be employed. For example, thespleen cells and myeloma cells may be combined with a nonionic detergentfor a few minutes and then plated at low density on a selective mediumthat supports the growth of hybrid cells, but not myeloma cells. Apreferred selection technique uses HAT (hypoxanthine, aminopterin,thymidine) selection. After a sufficient time, usually about 1 to 2weeks, colonies of hybrids are observed. Single colonies are selectedand their culture supernatants tested for binding activity against thepeptide. Hybridomas having high reactivity and specificity arepreferred.

Monoclonal antibodies may be isolated from the supernatants of growinghybridoma colonies. In addition, various techniques may be employed toenhance the yield, such as injection of the hybridoma cell line into theperitoneal cavity of a suitable vertebrate host, such as a mouse.Monoclonal antibodies may then be harvested from the ascites fluid orthe blood. Contaminants may be removed from the antibodies byconventional techniques, such as chromatography, gel filtration,precipitation, and extraction. The peptides of this invention may beused in the purification process in, for example, an affinitychromatography step.

Within certain embodiments, the use of antigen-binding fragments ofantibodies may be preferred. Such fragments include Fab fragments, whichmay be prepared using standard techniques. Briefly, immunoglobulins maybe purified from rabbit serum by affinity chromatography on Protein Abead columns (Harlow and Lane, 1988) and digested by papain to yield Faband Fc fragments. The Fab and Fc fragments may be separated by affinitychromatography on Protein A bead columns.

Monoclonal antibodies and fragments thereof may be coupled to one ormore therapeutic agents. Suitable agents in this regard includeradioactive tracers and chemotherapeutic agents, which may be used, forexample, to purge autologous bone marrow in vitro). Representativetherapeutic agents include radionuclides, differentiation inducers,drugs, toxins, and derivatives thereof. Preferred radionuclides include⁹⁰Y, ¹²³I, ¹²⁵I, ¹³¹I, ¹⁸⁶Re, ¹⁸⁸Re, ²¹¹At, and ²¹²Bi. Preferred drugsinclude methotrexate, and pyrimidine and purine analogs. Preferreddifferentiation inducers include phorbol esters and butyric acid.Preferred toxins include ricin, abrin, diptheria toxin, cholera toxin,gelonin, Pseudomonas exotoxin, Shigella toxin, and pokeweed antiviralprotein. For diagnostic purposes, coupling of radioactive agents may beused to facilitate tracing of metastases or to determine the location ofhematological malignancy related-positive tumors.

A therapeutic agent may be coupled (e.g., covalently bonded) to asuitable monoclonal antibody either directly or indirectly (e.g., via alinker group). A direct reaction between an agent and an antibody ispossible when each possesses a substituent capable of reacting with theother. For example, a nucleophilic group, such as an amino or sulfhydrylgroup, on one may be capable of reacting with a carbonyl-containinggroup, such as an anhydride or an acid halide, or with an alkyl groupcontaining a good leaving group (e.g., a halide) on the other.

Alternatively, it may be desirable to couple a therapeutic agent and anantibody via a linker group. A linker group can function as a spacer todistance an antibody from an agent in order to avoid interference withbinding capabilities. A linker group can also serve to increase thechemical reactivity of a substituent on an agent or an antibody, andthus increase the coupling efficiency. An increase in chemicalreactivity may also facilitate the use of agents, or functional groupson agents, which otherwise would not be possible.

It will be evident to those skilled in the art that a variety ofbifunctional or polyfunctional reagents, both homo- andhetero-functional (such as those described in the catalog of the PierceChemical Co., Rockford, Ill.), may be employed as the linker group.Coupling may be affected, for example, through amino groups, carboxylgroups, and sulfhydryl groups or oxidized carbohydrate residues. Thereare numerous references describing such methodology, e.g., U.S. Pat. No.4,671,958.

Where a therapeutic agent is more potent when free from the antibodyportion of the immunoconjugates of the present invention, it may bedesirable to use a linker group that is cleavable during or uponinternalization into a cell. A number of different cleavable linkergroups have been described. The mechanisms for the intracellular releaseof an agent from these linker groups include cleavage by reduction of adisulfide bond (U.S. Pat. No. 4,489,710), by irradiation of aphotolabile bond (U.S. Pat. No. 4,625,014), by hydrolysis of derivatizedamino acid side chains (U.S. Pat. No. 4,638,045), by serumcomplement-mediated hydrolysis (U.S. Pat. No. 4,671,958), andacid-catalyzed hydrolysis (U.S. Pat. No. 4,569,789).

It may be desirable to couple more than one agent to an antibody. In oneembodiment, multiple molecules of an agent are coupled to one antibodymolecule. In another embodiment, more than one type of agent may becoupled to one antibody. Regardless of the particular embodiment,immunoconjugates with more than one agent may be prepared in a varietyof ways. For example, more than one agent may be coupled directly to anantibody molecule, or linkers that provide multiple sites for attachmentcan be used. Alternatively, a carrier can be used. A carrier may bearthe agents in a variety of ways, including covalent bonding eitherdirectly or via a linker group. Suitable carriers include proteins suchas albumins (U.S. Pat. No. 4,507,234), peptides and polysaccharides suchas aminodextran (U.S. Pat. No. 4,699,784). A carrier may also bear anagent by noncovalent bonding or by encapsulation, such as within aliposome vesicle (U.S. Pat. No. 4,429,008 and U.S. Pat. No. 4,873,088).Carriers specific for radionuclide agents include radiohalogenated smallmolecules and chelating compounds. For example, U.S. Pat. No. 4,735,792discloses representative radiohalogenated small molecules and theirsynthesis. A radionuclide chelate may be formed from chelating compoundsthat include those containing nitrogen and sulfur atoms as the donoratoms for binding the metal, or metal oxide, radionuclide. For example,U.S. Pat. No. 4,673,562 discloses representative chelating compounds andtheir synthesis.

A variety of routes of administration for the antibodies andimmunoconjugates may be used. Typically, administration will beintravenous, intramuscular, subcutaneous or in the bed of a resectedtumor. It will be evident that the precise dose of theantibody/immunoconjugate will vary depending upon the antibody used, theantigen density on the tumor, and the rate of clearance of the antibody.

Also provided herein are anti-idiotypic antibodies that mimic animmunogenic portion of hematological malignancy related. Such antibodiesmay be raised against an antibody, or an antigen-binding fragmentthereof, that specifically binds to an immunogenic portion ofhematological malignancy related, using well-known techniques.Anti-idiotypic antibodies that mimic an immunogenic portion ofhematological malignancy related are those antibodies that bind to anantibody, or antigen-binding fragment thereof, that specifically bindsto an immunogenic portion of hematological malignancy related, asdescribed herein.

Irrespective of the source of the original hematological malignancyrelated peptide-specific antibody, the intact antibody, antibodymultimers, or any one of a variety of functional, antigen-bindingregions of the antibody may be used in the present invention. Exemplaryfunctional regions include scFv, Fv, Fab′, Fab and F(ab)₂ fragments ofthe hematological malignancy related peptide-specific antibodies.Techniques for preparing such constructs are well known to those in theart and are further exemplified herein.

The choice of antibody construct may be influenced by various factors.For example, prolonged half-life can result from the active readsorptionof intact antibodies within the kidney, a property of the Fc piece ofimmunoglobulin. IgG based antibodies, therefore, are expected to exhibitslower blood clearance than their Fab′ counterparts. However, Fab′fragment-based compositions will generally exhibit better tissuepenetrating capability.

Antibody fragments can be obtained by proteolysis of the wholeimmunoglobulin by the non-specific thiol protease, papain. Papaindigestion yields two identical antigen-binding fragments, termed “Fabfragments,” each with a single antigen-binding site, and a residual “Fcfragment.”

Papain should first be activated by reducing the sulfhydryl group in theactive site with cysteine, 2-mercaptoethanol or dithiothreitol. Heavymetals in the stock enzyme should be removed by chelation with EDTA (2mM) to ensure maximum enzyme activity. Enzyme and substrate are normallymixed together in the ratio of 1:100 by weight. After incubation, thereaction can be stopped by irreversible alkylation of the thiol groupwith iodoacetamide or simply by dialysis. The completeness of thedigestion should be monitored by SDS-PAGE and the various fractionsseparated by Protein A-Sepharose or ion exchange chromatography.

The usual procedure for preparation of F(ab′)₂ fragments from IgG ofrabbit and human origin is limited proteolysis by the enzyme pepsin. Theconditions, 100× antibody excess wt./wt. in acetate buffer at pH 4.5,37° C., suggest that antibody is cleaved at the C-terminal side of theinter-heavy-chain disulfide bond. Rates of digestion of mouse IgG mayvary with subclass and it may be difficult to obtain high yields ofactive F(ab′)₂ fragments without some undigested or completely degradedIgG. In particular, IgG_(2b) is highly susceptible to completedegradation. The other subclasses require different incubationconditions to produce optimal results, all of which is known in the art.

Pepsin treatment of intact antibodies yields an F(ab)₂ fragment that hastwo antigen-combining sites and is still capable of cross-liningantigen. Digestion of rat IgG by pepsin requires conditions includingdialysis in 0.1 M acetate buffer, pH 4.5, and then incubation for fourhrs with 1% wt./wt. pepsin; IgG₁ and IgG_(2a) digestion is improved iffirst dialyzed against 0.1 M formate buffer, pH 2.8, at 4° C., for 16hrs followed by acetate buffer. IgG_(2b) gives more consistent resultswith incubation in staphylococcal V8 protease (3% wt./wt.) in 0.1 Msodium phosphate buffer, pH 7.8, for four hrs at 37° C.

A Fab fragment also contains the constant domain of the light chain andthe first constant domain (CH1) of the heavy chain. Fab′ fragmentsdiffer from Fab fragments by the addition of a few residues at thecarboxyl terminus of the heavy chain CH1 domain including one or morecysteine(s) from the antibody hinge region. F(ab)₂ antibody fragmentswere originally produced as pairs of Fab′ fragments that have hingecysteines between them. Other chemical couplings of antibody fragmentsare also known.

The term “variable,” as used herein in reference to antibodies, meansthat certain portions of the variable domains differ extensively insequence among antibodies, and are used in the binding and specificityof each particular antibody to its particular antigen. However, thevariability is not evenly distributed throughout the variable domains ofantibodies. It is concentrated in three segments termed “hypervariableregions,” both in the light chain and the heavy chain variable domains.

The more highly conserved portions of variable domains are called theframework region (FR). The variable domains of native heavy and lightchains each comprise four FRs (FR1, FR2, FR3 and FR4, respectively),largely adopting a β-sheet configuration, connected by threehypervariable regions, which form loops connecting, and in some cases,forming part of, the β-sheet structure.

The hypervariable regions in each chain are held together in closeproximity by the FRs and, with the hypervariable regions from the otherchain, contribute to the formation of the antigen-binding site ofantibodies (Kabat et al., 1991, specifically incorporated herein byreference). The constant domains are not involved directly in binding anantibody to an antigen, but exhibit various effector functions, such asparticipation of the antibody in antibody-dependent cellular toxicity.

The term “hypervariable region,” as used herein, refers to the aminoacid residues of an antibody that are responsible for antigen-binding.The hypervariable region comprises amino acid residues from a“complementarity determining region” or “CDR” (i.e. residues 24-34 (L1),50-56 (L2) and 89-97 (L3) in the light chain variable domain and 31-35(H1), 50-56 (H2) and 95-102 (H3) in the heavy chain variable domain(Kabat et al., 1991, specifically incorporated herein by reference)and/or those residues from a “hypervariable loop” (i.e., residues 26-32(L1), 50-52 (L2) and 91-96 (L3) in the light chain variable domain and26-32 (H1), 53-55 (H2) and 96-101 (H3) in the heavy chain variabledomain). “Framework” or “FR” residues are those variable domain residuesother than the hypervariable region residues as herein defined.

An “Fv” fragment is the minimum antibody fragment that contains acomplete antigen-recognition and binding site. This region consists of adimer of one heavy chain and one light chain variable domain in tight,con-covalent association. It is in this configuration that threehypervariable regions of each variable domain interact to define anantigen-binding site on the surface of the V_(H)-V_(L) dimer.Collectively, six hypervariable regions confer antigen-bindingspecificity to the antibody. However, even a single variable domain (orhalf of an Fv comprising only three hypervariable regions specific foran antigen) has the ability to recognize and bind antigen, although at alower affinity than the entire binding site.

“Single-chain Fv” or “sFv” antibody fragments comprise the V_(H) andV_(L) domains of antibody, wherein these domains are present in a singlepolypeptide chain. Generally, the Fv polypeptide further comprises apolypeptide linker between the V_(H) and V_(L) domains that enables thesFv to form the desired structure for antigen binding.

“Diabodies” are small antibody fragments with two antigen-binding sites,which fragments comprise a heavy chain variable domain (V_(H)) connectedto a light chain variable domain (V_(L)) in the same polypeptide chain(V_(H)-V_(L)). By using a linker that is too short to allow pairingbetween the two domains on the same chain, the domains are forced topair with the complementary domains of another chain and create twoantigen-binding sites. Diabodies are described in European Pat. Appl.No. EP 404,097 and Intl. Pat. Appl. Publ. No. WO 93/11161, eachspecifically incorporated herein by reference. “Linear antibodies”,which can be bispecific or monospecific, comprise a pair of tandem Fdsegments (V_(H)-C_(H)1-V_(H)-C_(H)1) that form a pair of antigen bindingregions, as described in Zapata et al. (1995), specifically incorporatedherein by reference.

Other types of variants are antibodies with improved biologicalproperties relative to the parent antibody from which they aregenerated. Such variants, or second-generation compounds, are typicallysubstitutional variants involving one or more substituted hypervariableregion residues of a parent antibody. A convenient way for generatingsuch substitutional variants is affinity maturation using phage display.

In affinity maturation using phage display, several hypervariable regionsites (e.g., 6 to 7 sites) are mutated to generate all possible aminosubstitutions at each site. The antibody variants thus generated aredisplayed in a monovalent fashion from filamentous phage particles asfusions to the gene III product of M13 packaged within each particle.The phage-displayed variants are then screened for their biologicalactivity (e.g., binding affinity) as herein disclosed. In order toidentify candidate hypervariable region sites for modification,alanine-scanning mutagenesis can be performed on hypervariable regionresidues identified as contributing significantly to antigen binding.

Alternatively, or in addition, the crystal structure of theantigen-antibody complex be delineated and analyzed to identify contactpoints between the antibody and target. Such contact residues andneighboring residues are candidates for substitution. Once such variantsare generated, the panel of variants is subjected to screening, andantibodies with analogues but different or even superior properties inone or more relevant assays are selected for further development.

In using a Fab′ or antigen binding fragment of an antibody, with theattendant benefits on tissue penetration, one may derive additionaladvantages from modifying the fragment to increase its half-life. Avariety of techniques may be employed, such as manipulation ormodification of the antibody molecule itself, and also conjugation toinert carriers. Any conjugation for the sole purpose of increasinghalf-life, rather than to deliver an agent to a target, should beapproached carefully in that Fab′ and other fragments are chosen topenetrate tissues. Nonetheless, conjugation to non-protein polymers,such PEG and the like, is contemplated.

Modifications other than conjugation are therefore based upon modifyingthe structure of the antibody fragment to render it more stable, and/orto reduce the rate of catabolism in the body. One mechanism for suchmodifications is the use of D-amino acids in place of L-amino acids.Those of ordinary skill in the art will understand that the introductionof such modifications needs to be followed by rigorous testing of theresultant molecule to ensure that it still retains the desiredbiological properties. Further stabilizing modifications include the useof the addition of stabilizing moieties to either the N-terminal or theC-terminal, or both, which is generally used to prolong the half-life ofbiological molecules. By way of example only, one may wish to modify thetermini by acylation or amination.

Moderate conjugation-type modifications for use with the presentinvention include incorporating a salvage receptor binding epitope intothe antibody fragment. Techniques for achieving this include mutation ofthe appropriate region of the antibody fragment or incorporating theepitope as a peptide tag that is attached to the antibody fragment.Intl. Pat. Appl. Publ. No. WO 96/32478 is specifically incorporatedherein by reference for the purposes of further exemplifying suchtechnology. Salvage receptor binding epitopes are typically regions ofthree or more amino acids from one or two lops of the Fc domain that aretransferred to the analogous position on the antibody fragment. Thesalvage receptor-binding epitopes disclosed in Intl. Pat. Appl. Publ.No. WO 98/45331 are incorporated herein by reference for use with thepresent invention.

4.3 T Cell Compositions Specific for Hematological Malignancy-RelatedPeptides

Immunotherapeutic compositions may also, or alternatively, comprise Tcells specific for hematological malignancy related. Such cells maygenerally be prepared in vitro or ex vivo, using standard procedures.For example, T cells may be present within (or isolated from) bonemarrow, peripheral blood or a fraction of bone marrow or peripheralblood of a mammal, such as a patient, using a commercially availablecell separation system, such as the Isolex™ System, available fromNexell Therapeutics, Inc. (Irvine, Calif.; see also U.S. Pat. No.5,240,856; U.S. Pat. No. 5,215,926; Intl. Pat. Appl. Publ. No. WO89/06280; Intl. Pat. Appl. Publ. No. WO 91/16116 and Intl. Pat. Appl.Publ. No. WO 92/07243). Alternatively, T cells may be derived fromrelated or unrelated humans, non-human mammals, cell lines or cultures.

T cells may be stimulated with hematological malignancy related peptide,polynucleotide encoding a hematological malignancy related peptideand/or an antigen-presenting cell (APC) that expresses a hematologicalmalignancy related peptide. Such stimulation is performed underconditions and for a time sufficient to permit the generation of T cellsthat are specific for the hematological malignancy related peptide.Preferably, a hematological malignancy related peptide or polynucleotideis present within a delivery vehicle, such as a microsphere, tofacilitate the generation of antigen-specific T cells. Briefly, T cells,which may be isolated from a patient or a related or unrelated donor byroutine techniques (such as by Ficoll/Hypaque® density gradientcentrifugation of peripheral blood lymphocytes), are incubated withhematological malignancy related peptide. For example, T cells may beincubated in vitro for 2-9 days (typically 4 days) at 37° C. withhematological malignancy related peptide (e.g., 5 to 25 μg/ml) or cellssynthesizing a comparable amount of hematological malignancy relatedpeptide. It may be desirable to incubate a separate aliquot of a T cellsample in the absence of hematological malignancy related peptide toserve as a control.

T cells are considered to be specific for a hematological malignancyrelated peptide if the T cells kill target cells coated with ahematological malignancy related peptide or expressing a gene encodingsuch a peptide. T cell specificity may be evaluated using any of avariety of standard techniques. For example, within a chromium releaseassay or proliferation assay, a stimulation index of more than two foldincrease in lysis and/or proliferation, compared to negative controls,indicates T cell specificity. Such assays may be performed, for example,as described in Chen et al. (1994). Alternatively, detection of theproliferation of T cells may be accomplished by a variety of knowntechniques. For example, T cell proliferation can be detected bymeasuring an increased rate of DNA synthesis (e.g., by pulse-labelingcultures of T cells with tritiated thymidine and measuring the amount oftritiated thymidine incorporated into DNA). Other ways to detect T cellproliferation include measuring increases in interleukin-2 (IL-2)production, Ca²⁺ flux, or dye uptake, such as3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium. Alternatively,synthesis of lymphokines (such as interferon-gamma) can be measured orthe relative number of T cells that can respond to a hematologicalmalignancy related peptide may be quantified. Contact with ahematological malignancy related peptide (200 ng/ml-100 μg/ml,preferably 100 ng/ml-25 μg/ml) for 3-7 days should result in at least atwo-fold increase in proliferation of the T cells and/or contact asdescribed above for 2-3 hrs should result in activation of the T cells,as measured using standard cytokine assays in which a two-fold increasein the level of cytokine release (e.g., TNF or IFN-γ) is indicative of Tcell activation (Coligan et al., 1998). hematological malignancy relatedspecific T cells may be expanded using standard techniques. Withinpreferred embodiments, the T cells are derived from a patient or arelated or unrelated donor and are administered to the patient followingstimulation and expansion.

T cells that have been activated in response to a hematologicalmalignancy related peptide, polynucleotide or hematological malignancyrelated-expressing APC may be CD4⁺ and/or CD8⁺. Specific activation ofCD4⁺ or CD8⁺ T cells may be detected in a variety of ways. Methods fordetecting specific T cell activation include detecting the proliferationof T cells, the production of cytokines (e.g., lymphokines), or thegeneration of cytolytic activity (i.e., generation of cytotoxic T cellsspecific for hematological malignancy related). For CD4⁺ T cells, apreferred method for detecting specific T cell activation is thedetection of the proliferation of T cells. For CD8⁺ T cells, a preferredmethod for detecting specific T cell activation is the detection of thegeneration of cytolytic activity.

For therapeutic purposes, CD4⁺ or CD8⁺ T cells that proliferate inresponse to the hematological malignancy related peptide, polynucleotideor APC can be expanded in number either in vitro or in vivo.Proliferation of such T cells in vitro may be accomplished in a varietyof ways. For example, the T cells can be re-exposed to hematologicalmalignancy related peptide, with or without the addition of T cellgrowth factors, such as interleukin-2, and/or stimulator cells thatsynthesize a hematological malignancy related peptide. The addition ofstimulator cells is preferred where generating CD8⁺ T cell responses. Tcells can be grown to large numbers in vitro with retention ofspecificity in response to intermittent restimulation with hematologicalmalignancy related peptide. Briefly, for the primary in vitrostimulation (IVS), large numbers of lymphocytes (e.g., greater than4×10⁷) may be placed in flasks with media containing human serum.hematological malignancy related peptide (e.g., peptide at 10 μg/ml) maybe added directly, along with tetanus toxoid (e.g., 5 μg/ml). The flasksmay then be incubated (e.g., 37° C. for 7 days). For a second IVS, Tcells are then harvested and placed in new flasks with 2-3×10⁷irradiated peripheral blood mononuclear cells. hematological malignancyrelated peptide (e.g., 10 μg/ml) is added directly. The flasks areincubated at 37° C. for 7 days. On day 2 and day 4 after the second IVS,2-5 units of interleukin-2 (IL-2) may be added. For a third IVS, the Tcells may be placed in wells and stimulated with the individual's ownEBV transformed B cells coated with the peptide. IL-2 may be added ondays 2 and 4 of each cycle. As soon as the cells are shown to bespecific cytotoxic T cells, they may be expanded using a 10-daystimulation cycle with higher IL-2 (20 units) on days 2, 4 and 6.

Alternatively, one or more T cells that proliferate in the presence ofhematological malignancy related peptide can be expanded in number bycloning. Methods for cloning cells are well known in the art, andinclude limiting dilution. Responder T cells may be purified from theperipheral blood of sensitized patients by density gradientcentrifugation and sheep red cell rosetting and established in cultureby stimulating with the nominal antigen in the presence of irradiatedautologous filler cells. In order to generate CD4⁺ T cell lines,hematological malignancy related peptide is used as the antigenicstimulus and autologous peripheral blood lymphocytes (PBL) orlymphoblastoid cell lines (LCL) immortalized by infection with EpsteinBarr virus are used as antigen-presenting cells. In order to generateCD8⁺ T cell lines, autologous antigen-presenting cells transfected withan expression vector that produces hematological malignancy relatedpeptide may be used as stimulator cells. Established T cell lines may becloned 2-4 days following antigen stimulation by plating stimulated Tcells at a frequency of 0.5 cells per well in 96-well flat-bottom plateswith 1×10⁶ irradiated PBL or LCL cells and recombinant interleukin-2(rIL2) (50 U/ml). Wells with established clonal growth may be identifiedat approximately 2-3 weeks after initial plating and restimulated withappropriate antigen in the presence of autologous antigen-presentingcells, then subsequently expanded by the addition of low doses of rIL2(10 U/ml) 2-3 days following antigen stimulation. T cell clones may bemaintained in 24-well plates by periodic restimulation with antigen andrIL2 approximately every two weeks. Cloned and/or expanded cells may beadministered back to the patient as described, for example, by Chang etal., (1996).

Within certain embodiments, allogeneic T-cells may be primed (i.e.,sensitized to hematological malignancy related) in vivo and/or in vitro.Such priming may be achieved by contacting T cells with a hematologicalmalignancy related peptide, a polynucleotide encoding such a peptide ora cell producing such a peptide under conditions and for a timesufficient to permit the priming of T cells. In general, T cells areconsidered to be primed if, for example, contact with a hematologicalmalignancy related peptide results in proliferation and/or activation ofthe T cells, as measured by standard proliferation, chromium releaseand/or cytokine release assays as described herein. A stimulation indexof more than two fold increase in proliferation or lysis, and more thanthree fold increase in the level of cytokine, compared to negativecontrols indicates T-cell specificity. Cells primed in vitro may beemployed, for example, within bone marrow transplantation or as donorlymphocyte infusion.

T cells specific for hematological malignancy related can kill cellsthat express hematological malignancy related protein. Introduction ofgenes encoding T-cell receptor (TCR) chains for hematological malignancyrelated are used as a means to quantitatively and qualitatively improveresponses to hematological malignancy related bearing leukemia andcancer cells. Vaccines to increase the number of T cells that can reactto hematological malignancy related positive cells are one method oftargeting hematological malignancy related bearing cells. T cell therapywith T cells specific for hematological malignancy related is anothermethod. An alternative method is to introduce the TCR chains specificfor hematological malignancy related into T cells or other cells withlytic potential. In a suitable embodiment, the TCR alpha and beta chainsare cloned out from a hematological malignancy related specific T cellline and used for adoptive T cell therapy, such as described in WO96/30516, incorporated herein by reference.

4.4 Pharmaceutical Compositions and Vaccine Formulations

Within certain aspects, peptides, polynucleotides, antibodies and/or Tcells may be incorporated into pharmaceutical compositions orimmunogenic compositions (i.e., vaccines). Alternatively, apharmaceutical composition may comprise an antigen-presenting cell(e.g., a dendritic cell) transfected with a hematological malignancyrelated polynucleotide such that the antigen-presenting cell expresses ahematological malignancy related peptide. Pharmaceutical compositionscomprise one or more such compounds or cells and a physiologicallyacceptable carrier or excipient. Vaccines may comprise one or more suchcompounds or cells and an immunostimulant, such as an adjuvant or aliposome (into which the compound is incorporated). An immunostimulantmay be any substance that enhances or potentiates an immune response(antibody- and/or cell-mediated) to an exogenous antigen. Examples ofimmunostimulants include adjuvants, biodegradable microspheres (e.g.,polylactic galactide) and liposomes (into which the compound isincorporated) (U.S. Pat. No. 4,235,877). Vaccine preparation isgenerally described in, for example, Powell and Newman (1995).Pharmaceutical compositions and vaccines within the scope of the presentinvention may also contain other compounds, which may be biologicallyactive or inactive. For example, one or more immunogenic portions ofother tumor antigens may be present, either incorporated into a fusionpeptide or as a separate compound, within the composition or vaccine.

Within certain embodiments, pharmaceutical compositions and vaccines aredesigned to elicit T cell responses specific for a hematologicalmalignancy related peptide in a patient, such as a human. In general, Tcell responses may be favored through the use of relatively shortpeptides (e.g., comprising less than 23 consecutive amino acid residuesof a native hematological malignancy related peptide, preferably 4-16consecutive residues, more preferably 8-16 consecutive residues andstill more preferably 8-10 consecutive residues). Alternatively, or inaddition, a vaccine may comprise an immunostimulant that preferentiallyenhances a T cell response. In other words, the immunostimulant mayenhance the level of a T cell response to a hematological malignancyrelated peptide by an amount that is proportionally greater than theamount by which an antibody response is enhanced. For example, whencompared to a standard oil based adjuvant, such as CFA, animmunostimulant that preferentially enhances a T cell response mayenhance a proliferative T cell response by at least two fold, a lyticresponse by at least 10%, and/or T cell activation by at least two foldcompared to hematological malignancy related-negative control celllines, while not detectably enhancing an antibody response. The amountby which a T cell or antibody response to a hematological malignancyrelated peptide is enhanced may generally be determined using anyrepresentative technique known in the art, such as the techniquesprovided herein.

A pharmaceutical composition or vaccine may contain DNA encoding one ormore of the peptides as described above, such that the peptide isgenerated in situ. As noted above, the DNA may be present within any ofa variety of delivery systems known to those of ordinary skill in theart, including nucleic acid expression systems, bacterial and viralexpression systems and mammalian expression systems. Numerous genedelivery techniques are well known in the art (Rolland, 1998, andreferences cited therein). Appropriate nucleic acid expression systemscontain the necessary DNA, cDNA or RNA sequences for expression in thepatient (such as a suitable promoter and terminating signal). Bacterialdelivery systems involve the administration of a bacterium (such asBacillus-Calmette-Guerrin) that expresses an immunogenic portion of thepeptide on its cell surface or secretes such an epitope. In a preferredembodiment, the DNA may be introduced using a viral expression system(e.g., vaccinia or other pox virus, retrovirus, or adenovirus), whichmay involve the use of a non-pathogenic (defective), replicationcompetent virus (Fisher-Hoch et al., 1989; Flexner et al., 1989; Flexneret al., 1990; U.S. Pat. No. 4,603,112, U.S. Pat. No. 4,769,330, U.S.Pat. No. 5,017,487; Intl. Pat. Appl. Publ. No. WO 89/01973; U.S. Pat.No. 4,777,127; Great Britain Patent No. GB 2,200,651; European PatentNo. EP 0,345,242; Intl. Pat. Appl. Publ. No. WO 91/02805; Berkner, 1988;Rosenfeld et al., 1991; Kolls et al., 1994; Kass-Eisler et al, 1993;Guzman et al., 1993a; and Guzman et al., 1993). Techniques forincorporating DNA into such expression systems are well known to thoseof ordinary skill in the art. The DNA may also be “naked,” as described,for example, in Ulmer et al. (1993) and reviewed by Cohen (1993). Theuptake of naked DNA may be increased by coating the DNA ontobiodegradable beads, which are efficiently transported into the cells.It will be apparent that a vaccine may comprise both a polynucleotideand a peptide component. Such vaccines may provide for an enhancedimmune response.

As noted above, a pharmaceutical composition or vaccine may comprise anantigen-presenting cell that expresses a hematological malignancyrelated peptide. For therapeutic purposes, as described herein, theantigen-presenting cell is preferably an autologous dendritic cell. Suchcells may be prepared and transfected using standard techniques (Reeveset al., 1996; Tuting et al., 1998; and Nair et al., 1998). Expression ofa hematological malignancy related peptide on the surface of anantigen-presenting cell may be confirmed by in vitro stimulation andstandard proliferation as well as chromium release assays, as describedherein.

It will be apparent to those of ordinary skill in the art having thebenefit of the present teachings that a vaccine may containpharmaceutically acceptable salts of the polynucleotides and peptidesprovided herein. Such salts may be prepared from pharmaceuticallyacceptable non-toxic bases, including organic bases (e.g., salts ofprimary, secondary and tertiary amines and basic amino acids) andinorganic bases (e.g., sodium, potassium, lithium, ammonium, calcium andmagnesium salts). The phrases “pharmaceutically or pharmacologicallyacceptable” refer to molecular entities and compositions that do notproduce an adverse, allergic or other significant untoward reaction whenadministered to an animal, or a human, as appropriate. As used herein,“pharmaceutically acceptable carrier” includes any and all solvents,dispersion media, coatings, antibacterial and antifungal agents,isotonic and absorption delaying agents and the like. The use of suchmedia and agents for pharmaceutical active substances is well known inthe art. Except insofar as any conventional media or agent isincompatible with the active ingredient, its use in the therapeuticcompositions is contemplated. For human administration, preparationsshould meet sterility, pyrogenicity, and general safety and puritystandards as required by the Food and Drug Administration Office ofBiologics standards. Supplementary active ingredients can also beincorporated into the compositions.

While any suitable carrier known to those of ordinary skill in the artmay be employed in the pharmaceutical compositions of this invention,the type of carrier will vary depending on the mode of administration.Compositions of the present invention may be formulated for anyappropriate manner of administration, including for example, topical,oral, nasal, intravenous, intracranial, intraperitoneal, subcutaneous orintramuscular administration. For parenteral administration, such assubcutaneous injection, the carrier preferably comprises water, saline,alcohol, a fat, a wax or a buffer. For oral administration, any of theabove carriers or a solid carrier, such as mannitol, lactose, starch,magnesium stearate, sodium saccharine, talcum, cellulose, glucose,sucrose, and magnesium carbonate, may be employed. Biodegradablemicrospheres (e.g., polylactate polyglycolate) may also be employed ascarriers for the pharmaceutical compositions of this invention. Suitablebiodegradable microspheres are disclosed, for example, in U.S. Pat. Nos.4,897,268; 5,075,109; 5,928,647; 5,811,128; 5,820,883; 5,853,763;5,814,344 and 5,942,252. For certain topical applications, formulationas a cream or lotion, using well-known components, is preferred.

Such compositions may also comprise buffers (e.g., neutral bufferedsaline or phosphate buffered saline), carbohydrates (e.g., glucose,mannose, sucrose or dextrans), mannitol, proteins, peptides or aminoacids such as glycine, antioxidants, bacteriostats, chelating agentssuch as EDTA or glutathione, adjuvants (e.g., aluminum hydroxide),solutes that render the formulation isotonic, hypotonic or weaklyhypertonic with the blood of a recipient, suspending agents, thickeningagents and/or preservatives. Alternatively, compositions of the presentinvention may be formulated as a lyophilizate, or formulated with one ormore liposomes, microspheres, nanoparticles, or micronized deliverysystems using well-known technology.

Any of a variety of immunostimulants, such as adjuvants, may be employedin the preparation of vaccine compositions of this invention. Mostadjuvants contain a substance designed to protect the antigen from rapidcatabolism, such as aluminum hydroxide or mineral oil, and a stimulatorof immune responses, such as lipid A, Bortadella pertussis orMycobacterium tuberculosis derived proteins. Suitable adjuvants arecommercially available as, for example, alum-based adjuvants (e.g.,Alhydrogel, Rehydragel, aluminum phosphate, Algammulin, aluminumhydroxide); oil based adjuvants (Freund's Incomplete Adjuvant andComplete Adjuvant (Difco Laboratories, Detroit, Mich.), Specol, RIBI,TiterMax, Montanide ISA50 or Seppic MONTANIDE ISA 720); nonionic blockcopolymer-based adjuvants, cytokines (e.g., GM-CSF or Flat3-ligand);Merck Adjuvant 65 (Merck and Company, Inc., Rahway, N.J.); AS-2(SmithKline Beecham, Philadelphia, Pa.); salts of calcium, iron or zinc;an insoluble suspension of acylated tyrosine; acylated sugars;cationically or anionically derivatized polysaccharides;polyphosphazenes; biodegradable microspheres; monophosphoryl lipid A andQuil A. Cytokines, such as GM-CSF or interleukin-2, -7, or -12, may alsobe used as adjuvants.

Hemocyanins and hemoerythrins may also be used in the invention. The useof hemocyanin from keyhole limpet (KLH) is particularly preferred,although other molluscan and arthropod hemocyanins and hemoerythrins maybe employed. Various polysaccharide adjuvants may also be used.Polyamine varieties of polysaccharides are particularly preferred, suchas chitin and chitosan, including deacetylated chitin.

A further preferred group of adjuvants are the muramyl dipeptide (MDP,N-acetylmuramyl-L-alanyl-D-isoglutamine) group of bacterialpeptidoglycans. Derivatives of muramyl dipeptide, such as the amino acidderivative threonyl-MDP, and the fatty acid derivative MTPPE, are alsocontemplated.

U.S. Pat. No. 4,950,645 describes a lipophilic disaccharide-tripeptidederivative of muramyl dipeptide that is proposed for use in artificialliposomes formed from phosphatidyl choline and phosphatidyl glycerol. Itis said to be effective in activating human monocytes and destroyingtumor cells, but is non-toxic in generally high doses. The compounds ofU.S. Pat. No. 4,950,645, and Intl. Pat. Appl. Publ. No. WO 91/16347 arealso proposed for use in achieving particular aspects of the presentinvention.

BCG and BCG-cell wall skeleton (CWS) may also be used as adjuvants inthe invention, with or without trehalose dimycolate. Trehalosedimycolate may be used itself. Azuma et al. (1988) show that trehalosedimycolate administration correlates with augmented resistance toinfluenza virus infection in mice. Trehalose dimycolate may be preparedas described in U.S. Pat. No. 4,579,945.

Amphipathic and surface-active agents, e.g., saponin and derivativessuch as QS21 (Cambridge Biotech), form yet another group of preferredadjuvants for use with the immunogens of the present invention. Nonionicblock copolymer surfactants (Rabinovich et al., 1994; Hunter et al.,1991) may also be employed. Oligonucleotides, as described by Yamamotoet al. (1988) are another useful group of adjuvants. Quil A and lentinenare also preferred adjuvants.

Superantigens are also contemplated for use as adjuvants in the presentinvention. “Superantigens” are generally bacterial products thatstimulate a greater proportion of T lymphocytes than peptide antigenswithout a requirement for antigen processing (Mooney et. al., 1994).Superantigens include Staphylococcus exoproteins, such as the α, β, γand δ enterotoxins from S. aureus and S. epidermidis, and the α, β, γand δ E. coli exotoxins.

Common Staphylococcus enterotoxins are known as staphylococcalenterotoxin A (SEA) and staphylococcal enterotoxin B (SEB), withenterotoxins through E (SEE) being described (Rott et. al., 1992).Streptococcus pyogenes B (SEB), Clostridium perfringens enterotoxin(Bowness et. al., 1992), cytoplasmic membrane-associated protein (CAP)from S. pyogenes (Sato et. al., 1994) and toxic shock syndrome toxin-1(TSST-1) from S. aureus (Schwab et. al., 1993) are further usefulsuperantigens.

One group of adjuvants particularly preferred for use in the inventionare the detoxified endotoxins, such as the refined detoxified endotoxinof U.S. Pat. No. 4,866,034. These refined detoxified endotoxins areeffective in producing adjuvant responses in mammals.

The detoxified endotoxins may be combined with other adjuvants.Combination of detoxified endotoxins with trehalose dimycolate iscontemplated, as described in U.S. Pat. No. 4,435,386. Combinations ofdetoxified endotoxins with trehalose dimycolate and endotoxicglycolipids is also contemplated (U.S. Pat. No. 4,505,899), as iscombination of detoxified endotoxins with cell wall skeleton (CWS) orCWS and trehalose dimycolate, as described in U.S. Pat. Nos. 4,436,727,4,436,728 and 4,505,900. Combinations of just CWS and trehalosedimycolate, without detoxified endotoxins are also envisioned to beuseful, as described in U.S. Pat. No. 4,520,019.

MPL is currently one preferred immunopotentiating agent for use herein.References that concern the uses of MPL include Tomai et al. (1987),Chen et al. (1991) and Garg and Subbarao (1992), that each concerncertain roles of MPL in the reactions of aging mice; Elliott et al.(1991), that concerns the D-galactosamine loaded mouse and its enhancedsensitivity to lipopolysaccharide and MPL; Chase et al. (1986), thatrelates to bacterial infections; and Masihi et al. (1988), thatdescribes the effects of MPL and endotoxin on resistance of mice toToxoplasma gondii. Fitzgerald (1991) also reported on the use of MPL toup-regulate the immunogenicty of a syphilis vaccine and to confersignificant protection against challenge infection in rabbits.

Thus MPL is known to be safe for use, as shown in the above modelsystems. Phase-I clinical trials have also shown MPL to be safe for use(Vosika et al., 1984). Indeed, 100 μg/m² is known to be safe for humanuse, even on an outpatient basis (Vosika et al., 1984).

MPL generally induces polyclonal B cell activation (Baker et al., 1994),and has been shown to augment antibody production in many systems, forexample, in immunologically immature mice (Baker et al., 1988); in agingmice (Tomai and Johnson, 1989); and in nude and Xid mice (Madonna andVogel, 1986; Myers et al., 1995). Antibody production has been shownagainst erythrocytes (Hraba et al., 1993); T cell dependent andindependent antigens; Pnu-immune vaccine (Garg and Subbarao, 1992);isolated tumor-associated antigens (U.S. Pat. No. 4,877,611); againstsyngeneic tumor cells (Livingston et al., 1985; Ravindranath et al.,1994a;b); and against tumor-associated gangliosides (Ravindranath etal., 1994a;b).

Another useful attribute of MPL is that is augments IgM responses, asshown by Baker et al. (1988a), who describe the ability of MPL toincrease antibody responses in young mice. This is a particularly usefulfeature of an adjuvant for use in certain embodiments of the presentinvention. Myers et al. (1995) recently reported on the ability of MPLto induce IgM antibodies, by virtue T cell-independent antibodyproduction.

In the Myers et al. (1995) studies, MPL was conjugated to the hapten,TNP. MPL was proposed for use as a carrier for other haptens, such aspeptides.

MPL also activates and recruits macrophages (Verma et al., 1992). Tomaiand Johnson (1989) showed that MPL-stimulated T cells enhance IL-1secretion by macrophages. MPL is also known to activate superoxideproduction, lysozyme activity, phagocytosis, and killing of Candida inmurine peritoneal macrophages (Chen et al., 1991).

The effects of MPL on T cells include the endogenous production ofcytotoxic factors, such as TNF, in serum of BCG-primed mice by MPL(Bennett et al., 1988). Kovach et al. (1990) and Elliot et al. (1991)also show that MPL induces TNF activity. MPL is known to act with TNF-αto induce release of IFN-γ by NK cells. IFN-γ production by T cells inresponse to MPL was also documented by Tomai and Johnson (1989), andOdean et al. (1990).

MPL is also known to be a potent T cell adjuvant. For example, MPLstimulates proliferation of melanoma-antigen specific CTLs (Mitchell etal., 1988, 1993). Further, Baker et al. (1988b) showed that nontoxic MPLinactivated suppressor T cell activity. Naturally, in the physiologicalenvironment, the inactivation of T suppressor cells allows for increasedbenefit for the animal, as realized by, e.g., increased antibodyproduction. Johnson and Tomai (1988) have reported on the possiblecellular and molecular mediators of the adjuvant action of MPL.

MPL is also known to induce aggregation of platelets and tophosphorylate a platelet protein prior to induction of serotoninsecretion (Grabarek et al., 1990). This study shows that MPL is involvedin protein kinase C activation and signal transduction.

Many articles concern the structure and function of MPL include. Theseinclude Johnson et al. (1990), that describes the structuralcharacterization of MPL homologs obtained from Salmonella minnesotaRe595 lipopolysaccharide. The work of Johnson et al. (1990), in commonwith Grabarek et al. (1990), shows that the fatty acid moieties of MPLcan vary, even in commercial species. In separating MPL into eightfractions by thin layer chromatography, Johnson et al. (1990) found thatthree were particularly active, as assessed using human plateletresponses. The chemical components of the various MPL species werecharacterized by Johnson et al. (1990).

Baker et al. (1992) further analyzed the structural features thatinfluence the ability of lipid A and its analogs to abolish expressionof suppressor T cell activity. They reported that decreasing the numberof phosphate groups in lipid A from two to one (i.e., creatingmonophosphoryl lipid A, MPL) as well as decreasing the fatty acylcontent, primarily by removing the residue at the 3 position, resultedin a progressive reduction in toxicity; however, these structuralmodifications did not influence its ability to abolish the expression ofTs function (Baker et al., 1992). These types of MPL are ideal for usein the present invention.

Baker et al. (1992) also showed that reducing the fatty acyl contentfrom five to four (lipid A precursor IV_(A) or I_(a)) eliminated thecapacity to influence Ts function but not to induce polyclonalactivation of B cells. These studies show that in order to be able toabolish the expression of Ts function, lipid A must be a glucosaminedisaccharide; may have either one or two phosphate groups; and must haveat least five fatty acyl groups. Also, the chain length of thenonhydroxylated fatty acid, as well as the location of acyloxyacylgroups (2′ versus 3′ position), may play an important role (Baker etal., 1992).

In examining the relationship between chain length and position of fattyacyl groups on the ability of lipid A to abolish the expression ofsuppressor T-cell (Ts) activity, Baker et al. (1994) found that fattyacyl chain lengths of C₁₂ to C₁₄ appeared to be optimal for bioactivity.Therefore, although their use is still possible, lipid A preparationswith fatty acyl groups of relatively short chain length (C₁₀ to C₁₂ fromPseudomonas aeruginosa and Chromobacterium violaceum) or predominantlylong chain length (C₁₈ from Helicobacter pylori) are less preferred foruse in this invention.

Baker et al. (1994) also showed that the lipid A proximal inner coreregion oligosaccharides of some bacterial lipopolysaccharides increasethe expression of Ts activity; due mainly to the capacity of sucholigosaccharides, which are relatively conserved in structure amonggram-negative bacterial, to enlarge or expand upon the population ofCD8⁺ Ts generated during the course of a normal antibody response tounrelated microbial antigens. The minimal structure required for theexpression of the added immunosuppression observed was reported to be ahexasaccharide containing one 2-keto-3-deoxyoctonate residue, twoglucose residues, and three heptose residues to which are attached twopyrophosphorylethanolamine groups (Baker et al., 1994). This informationmay be considered in utilizing or even designing further adjuvants foruse in the invention.

In a generally related line of work, Tanamoto et al. (1994a;b; 1995)described the dissociation of endotoxic activities in a chemicallysynthesized Lipid A precursor after acetylation or succinylation. Thus,compounds such as “acetyl 406” and “succinyl 516” (Tanamoto et al.,1994a;b; 1995) are also contemplated for use in the invention

Synthetic MPLs form a particularly preferred group of antigens. Forexample, Brade et al. (1993) described an artificial glycoconjugatecontaining the bisphosphorylated glucosamine disaccharide backbone oflipid A that binds to anti-Lipid A MAbs. This is one candidate for usein certain aspects of the invention.

The MPL derivatives described in U.S. Pat. No. 4,987,237 areparticularly contemplated for use in the present invention. U.S. Pat.No. 4,987,237 describes MPL derivatives that contain one or more freegroups, such as amines, on a side chain attached to the primary hydroxylgroups of the monophosphoryl lipid A nucleus through an ester group. Thederivatives provide a convenient method for coupling the lipid A throughcoupling agents to various biologically active materials. Theimmunostimulant properties of lipid A are maintained. All MPLderivatives in accordance with U.S. Pat. No. 4,987,237 are envisionedfor use in the MPL adjuvant-incorporated cells of this invention.

Various adjuvants, even those that are not commonly used in humans, maystill be employed in animals, where, for example, one desires to raiseantibodies or to subsequently obtain activated T cells. The toxicity orother adverse effects that may result from either the adjuvant or thecells, e.g., as may occur using non-irradiated tumor cells, isirrelevant in such circumstances.

Within the vaccines provided herein, the adjuvant composition ispreferably designed to induce an immune response predominantly of theTh1 type. High levels of Th1-type cytokines (e.g., IFN-γ, TNFα, IL-2 andIL-12) tend to favor the induction of cell-mediated immune responses toan administered antigen. In contrast, high levels of Th2-type cytokines(e.g., IL-4, IL-5, IL-6 and IL-10) tend to favor the induction ofhumoral immune responses. Following application of a vaccine as providedherein, a patient will support an immune response that includes Th1- andTh2-type responses. Within a preferred embodiment, in which a responseis predominantly Th1-type, the level of Th1-type cytokines will increaseto a greater extent than the level of Th2-type cytokines. The levels ofthese cytokines may be readily assessed using standard assays. For areview of the families of cytokines see e.g., Mosmann and Coffman(1989).

Preferred adjuvants for use in eliciting a predominantly Th1-typeresponse include, for example, a combination of monophosphoryl lipid A,preferably 3-de-O-acylated monophosphoryl lipid A (3D-MPL), togetherwith an aluminum salt. MPL adjuvants are available from CorixaCorporation (Seattle, Wash.; see e.g., U.S. Pat. Nos. 4,436,727;4,877,611; 4,866,034 and 4,912,094, each of which is specificallyincorporated herein by reference in its entirety). CpG-containingoligonucleotides (in which the CpG dinucleotide is unmethylated) alsoinduce a predominantly Th1 response. Such oligonucleotides are wellknown and are described, for example, in Intl. Pat. Appl. Publ. No. WO96/02555 and Intl. Pat. Appl. Publ. No. WO 99/33488. ImmunostimulatoryDNA sequences are also described, for example, by Sato et al. (1996).Another preferred adjuvant is a saponin, preferably QS21 (AquilaBiopharmaceuticals Inc., Framingham, Mass.), which may be used alone orin combination with other adjuvants. For example, an enhanced systeminvolves the combination of a monophosphoryl lipid A and saponinderivative, such as the combination of QS21 and 3D-MPL (see e.g., Intl.Pat. Appl. Publ. No. WO 94/00153), or a less reactogenic compositionwhere the QS21 is quenched with cholesterol (see e.g., Intl. Pat. Appl.Publ. No. WO 96/33739). Other preferred formulations comprise anoil-in-water emulsion and tocopherol. A particularly potent adjuvantformulation involving QS21, 3D-MPL and tocopherol in an oil-in-wateremulsion has also been described (see e.g., Intl. Pat. Appl. Publ. No.WO 95/17210).

Other preferred adjuvants include Montanide ISA 720 (Seppic), SAF(Chiron), ISCOMS (CSL), MF-59 (Chiron), the SBAS series of adjuvants(e.g., SBAS-2 or SBAS-4, available from SmithKline Beecham, Rixensart,Belgium), Detox (Corixa Corporation), RC-529 (Corixa Corporation) andaminoalkyl glucosaminide 4-phosphates (AGPs).

Any vaccine provided herein may be prepared using well-known methodsthat result in a combination of one or more antigens, one or moreimmunostimulants or adjuvants and one or more suitable carriers,excipients, or pharmaceutically acceptable buffers. The compositionsdescribed herein may be administered as part of a sustained releaseformulation (i.e., a formulation such as a capsule, sponge or gel[composed of polysaccharides, for example] that effects a slow releaseof compound following administration). Such formulations may generallybe prepared using well-known technology (Coombes et al., 1996) andadministered by, for example, oral, rectal or subcutaneous implantation,or by implantation at the desired target site. Sustained-releaseformulations may contain a peptide, polynucleotide or antibody dispersedin a carrier matrix and/or contained within a reservoir surrounded by arate-controlling membrane.

Carriers for use within such formulations are preferably biocompatible,and may also be biodegradable; preferably the formulation provides arelatively constant level of active component release. Such carriersinclude microparticles of poly(lactide-co-glycolide), as well aspolyacrylate, latex, starch, cellulose and dextran. Otherdelayed-release carriers include supramolecular biovectors, whichcomprise a non-liquid hydrophilic core (e.g. a cross-linkedpolysaccharide or oligosaccharide) and, optionally, an external layercomprising an amphiphilic compound, such as a phospholipid (U.S. Pat.No. 5,151,254; Intl. Pat. Appl. Publ. No. WO 94/20078; Intl. Pat. Appl.Publ. No. WO/94/23701; and Intl. Pat. Appl. Publ. No. WO 96/06638). Theamount of active compound contained within a sustained releaseformulation depends upon the site of implantation, the rate and expectedduration of release and the nature of the condition to be treated orprevented.

Any of a variety of delivery vehicles may be employed withinpharmaceutical compositions and vaccines to facilitate production of anantigen-specific immune response that targets tumor cells. Deliveryvehicles include antigen-presenting cells (APCs), such as dendriticcells, macrophages, B cells, monocytes and other cells that may beengineered to be efficient APCs. Such cells may, but need not, begenetically modified to increase the capacity for presenting theantigen, to improve activation and/or maintenance of the T cellresponse, to have anti-tumor effects per se and/or to be immunologicallycompatible with the receiver (i.e., matched HLA haplotype). APCs maygenerally be isolated from any of a variety of biological fluids andorgans, including tumor and peritumoral tissues, and may be autologous,allogeneic, syngeneic or xenogeneic cells.

Certain preferred embodiments of the present invention use dendriticcells or progenitors thereof as antigen-presenting cells. Dendriticcells are highly potent APCs (Banchereau and Steinman, 1998) and havebeen shown to be effective as a physiological adjuvant for elicitingprophylactic or therapeutic antitumor immunity (Timmerman and Levy,1999). In general, dendritic cells may be identified based on theirtypical shape (stellate in situ, with marked cytoplasmic processes(dendrites) visible in vitro), their ability to take up, process andpresent antigens with high efficiency and their ability to activatenaive T cell responses. Dendritic cells may, of course, be engineered toexpress specific cell-surface receptors or ligands that are not commonlyfound on dendritic cells in vivo or ex vivo, and such modified dendriticcells are contemplated by the present invention. As an alternative todendritic cells, secreted vesicles antigen-loaded dendritic cells(called exosomes) may be used within a vaccine (Zitvogel et al., 1998).

Dendritic cells and progenitors may be obtained from peripheral blood,bone marrow, tumor-infiltrating cells, peritumoral tissues-infiltratingcells, lymph nodes, spleen, skin, umbilical cord blood or any othersuitable tissue or fluid. For example, dendritic cells may bedifferentiated ex vivo by adding a combination of cytokines such asGM-CSF, IL-4, IL-13 and/or TNFα to cultures of monocytes harvested fromperipheral blood. Alternatively, CD34 positive cells harvested fromperipheral blood, umbilical cord blood or bone marrow may bedifferentiated into dendritic cells by adding to the culture mediumcombinations of GM-CSF, IL-3, TNFα, CD40 ligand, LPS, flt3 ligand and/orother compound(s) that induce differentiation, maturation andproliferation of dendritic cells.

Dendritic cells are conveniently categorized as “immature” and “mature”cells, which allows a simple way to discriminate between two wellcharacterized phenotypes. However, this nomenclature should not beconstrued to exclude all possible intermediate stages ofdifferentiation. Immature dendritic cells are characterized as APC witha high capacity for antigen uptake and processing, which correlates withthe high expression of Fcγ receptor and mannose receptor. The maturephenotype is typically characterized by a lower expression of thesemarkers, but a high expression of cell surface molecules responsible forT cell activation such as class I and class II MHC, adhesion molecules(e.g., CD54 and CD11) and costimulatory molecules (e.g., CD40, CD80,CD86 and 4-1BB).

APCs may generally be transfected with a polynucleotide encoding ahematological malignancy related peptide, such that the peptide, or animmunogenic portion thereof, is expressed on the cell surface. Suchtransfection may take place ex vivo, and a composition or vaccinecomprising such transfected cells may then be used for therapeuticpurposes, as described herein. Alternatively, a gene delivery vehiclethat targets a dendritic or other antigen-presenting cell may beadministered to a patient, resulting in transfection that occurs invivo. In vivo and ex vivo transfection of dendritic cells, for example,may generally be performed using any methods known in the art, such asthose described in Intl. Pat. Appl. Publ. No. WO 97/24447, or the genegun approach described by Mahvi et al. (1997). Antigen loading ofdendritic cells may be achieved by incubating dendritic cells orprogenitor cells with the hematological malignancy related peptide, DNA(naked or within a plasmid vector) or RNA; or with antigen-expressingrecombinant bacterium or viruses (e.g., vaccinia, fowlpox, adenovirus orlentivirus vectors). Prior to loading, the peptide may be covalentlyconjugated to an immunological partner that provides T cell help (e.g.,a carrier molecule). Alternatively, a dendritic cell may be pulsed witha non-conjugated immunological partner, separately or in the presence ofthe peptide.

Combined therapeutics is also contemplated, and the same type ofunderlying pharmaceutical compositions may be employed for both singleand combined medicaments. Vaccines and pharmaceutical compositions maybe presented in unit-dose or multi-dose containers, such as sealedampoules or vials. Such containers are preferably hermetically sealed topreserve sterility of the formulation until use. In general,formulations may be stored as suspensions, solutions or emulsions inoily or aqueous vehicles. Alternatively, a vaccine or pharmaceuticalcomposition may be stored in a freeze-dried condition requiring only theaddition of a sterile liquid carrier immediately prior to use.

4.5 Diagnostic and Prognostic Methods for Hematological MalignancyDiseases

The present invention further provides methods for detecting a malignantdisease associated with one or more of the polypeptide or polynucleotidecompositions disclosed herein, and for monitoring the effectiveness ofan immunization or therapy for such a disease. To determine the presenceor absence of a malignant disease associated with one or more of thepolypeptide or polynucleotide compositions disclosed herein, a patientmay be tested for the level of T cells specific for one or more of suchcompositions. Within certain methods, a biological sample comprisingCD4⁺ and/or CD8⁺ T cells isolated from a patient is incubated with oneor more of the polypeptide or polynucleotide compositions disclosedherein, and/or an APC that expresses one or more of such peptides orpolypeptides, and the presence or absence of specific activation of theT cells is detected, as described herein. Suitable biological samplesinclude, but are not limited to, isolated T cells. For example, T cellsmay be isolated from a patient by routine techniques (such as byFicoll/Hypaque density gradient centrifugation of peripheral bloodlymphocytes). T cells may be incubated in vitro for 2-9 days (typically4 days) at 37° C. with one or more of the disclosed peptide, polypeptideor polynucleotide compositions (e.g., 5-25 μg/ml). It may be desirableto incubate another aliquot of a T cell sample in the absence of thecomposition to serve as a control. For CD4⁺ T cells, activation ispreferably detected by evaluating proliferation of the T cells. For CD8⁺T cells, activation is preferably detected by evaluating cytolyticactivity. A level of proliferation that is at least two fold greaterand/or a level of cytolytic activity that is at least 20% greater thanin disease-free patients indicates the presence of a malignant diseaseassociated with expression or one or more of the disclosed polypeptideor polynucleotide compositions. Further correlation may be made, usingmethods well known in the art, between the level of proliferation and/orcytolytic activity and the predicted response to therapy. In particular,patients that display a higher antibody, proliferative and/or lyticresponse may be expected to show a greater response to therapy.

Within other methods, a biological sample obtained from a patient istested for the level of antibody specific for one or more of thehematological malignancy-related peptides or polypeptide s disclosedherein. The biological sample is incubated with hematologicalmalignancy-related peptide or polypeptide, or a polynucleotide encodingsuch a peptide or polypeptide, and/or an APC that expresses such apeptide or polypeptide under conditions and for a time sufficient toallow immunocomplexes to form. Immunocomplexes formed between theselected peptide or polypeptide and antibodies in the biological samplethat specifically bind to the selected peptide or polypeptide are thendetected. A biological sample for use within such methods may be anysample obtained from a patient that would be expected to containantibodies. Suitable biological samples include blood, sera, ascites,bone marrow, pleural effusion, and cerebrospinal fluid.

The biological sample is incubated with the selected peptide orpolypeptide in a reaction mixture under conditions and for a timesufficient to permit immunocomplexes to form between the selectedpeptide or polypeptide and antibodies that are immunospecific for such apeptide or polypeptide. For example, a biological sample and a selectedpeptide or polypeptide peptide may be incubated at 4° C. for 24-48 hrs.

Following the incubation, the reaction mixture is tested for thepresence of immuno-complexes. Detection of immunocomplexes formedbetween the selected peptide or polypeptide and antibodies present inthe biological sample may be accomplished by a variety of knowntechniques, such as radioimmunoassays (RIA) and enzyme linkedimmunosorbent assays (ELISA). Suitable assays are well known in the artand are amply described in the scientific and patent literature (Harlowand Lane, 1988). Assays that may be used include, but are not limitedto, the double monoclonal antibody sandwich immunoassay technique (U.S.Pat. No. 4,376,110); monoclonal-polyclonal antibody sandwich assays(Wide et al., 1970); the “western blot” method (U.S. Pat. No.4,452,901); immunoprecipitation of labeled ligand (Brown et al., 1980);enzyme-linked immunosorbent assays (Raines and Ross, 1982);immunocytochemical techniques, including the use of fluorochromes(Brooks et al., 1980); and neutralization of activity (Bowen-Pope etal., 1984). Other immunoassays include, but are not limited to, thosedescribed in U.S. Pat. Nos. 3,817,827; 3,850,752; 3,901,654; 3,935,074;3,984,533; 3,996,345; 4,034,074; and 4,098,876.

For detection purposes, the selected peptide or polypeptide may eitherbe labeled or unlabeled. Unlabeled polypeptide peptide may be used inagglutination assays or in combination with labeled detection reagentsthat bind to the immunocomplexes (e.g., anti-immunoglobulin, protein G,Protein A or a lectin and secondary antibodies, or antigen-bindingfragments thereof, capable of binding to the antibodies thatspecifically bind to the selected hematological malignancy-relatedpeptide or polypeptide). If the selected peptide or polypeptide islabeled, the reporter group may be any suitable reporter group known inthe art, including radioisotopes, fluorescent groups, luminescentgroups, enzymes, biotin and dye particles.

Within certain assays, unlabeled peptide or polypeptide is immobilizedon a solid support. The solid support may be any material known to thoseof ordinary skill in the art to which the peptide may be attached. Forexample, the solid support may be a test well in a microtiter plate or anitrocellulose or other suitable membrane. Alternatively, the supportmay be a bead or disc, such as glass, fiberglass, latex or a plasticmaterial such as polystyrene or polyvinylchloride. The support may alsobe a magnetic particle or a fiber optic sensor, such as those disclosed,for example, in U.S. Pat. No. 5,359,681. The peptide may be immobilizedon the solid support using a variety of techniques known to those ofskill in the art, which are amply described in the patent and scientificliterature. In the context of the present invention, the term“immobilization” refers to both noncovalent association, such asadsorption, and covalent attachment (which may be a direct linkagebetween the antigen and functional groups on the support or may be alinkage by way of a cross-linking agent). Immobilization by adsorptionto a well in a microtiter plate or to a membrane is preferred. In suchcases, adsorption may be achieved by contacting the selected peptide orpolypeptide, in a suitable buffer, with the solid support for a suitableamount of time. The contact time varies with temperature, but istypically between about 1 hour and about 1 day. In general, contacting awell of a plastic microtiter plate (such as polystyrene orpolyvinylchloride) with an amount of peptide ranging from about 10 ng toabout 10 μg, and preferably about 100 ng to about 1 μg, is sufficient toimmobilize an adequate amount of peptide.

Following immobilization, the remaining protein binding sites on thesupport are typically blocked. Any suitable blocking agent known tothose of ordinary skill in the art, such as bovine serum albumin, Tween™20™ (Sigma Chemical Co., St. Louis, Mo.), heat-inactivated normal goatserum (NGS), or BLOTTO (buffered solution of nonfat dry milk which alsocontains a preservative, salts, and an antifoaming agent) may be used.The support is then incubated with a biological sample suspected ofcontaining specific antibody. The sample can be applied neat, or, moreoften, it can be diluted, usually in a buffered solution which containsa small amount (0.1%-5.0% by weight) of protein, such as BSA, NGS, orBLOTTO. In general, an appropriate contact time (i.e., incubation time)is a period of time that is sufficient to detect the presence ofantibody or an antigen binding fragment that is immunospecific for theselected peptide or polypeptide within a sample containing such anantibody or binding fragment thereof. Preferably, the contact time issufficient to achieve a level of binding that is at least about 95% ofthat achieved at equilibrium between bound and unbound antibody orantibody fragment. Those of ordinary skill in the art will recognizethat the time necessary to achieve equilibrium may be readily determinedby assaying the level of binding that occurs over a period of time. Atroom temperature, an incubation time of about 30 min is generallysufficient.

Unbound sample may then be removed by washing the solid support with anappropriate buffer, such as PBS containing 0.1% Tween™ 20. A detectionreagent that binds to the immunocomplexes and that comprises at least afirst detectable label or “reporter” molecule may then be added. Thedetection reagent is incubated with the immunocomplex for an amount oftime sufficient to detect the bound antibody or antigen binding fragmentthereof. An appropriate amount of time may generally be determined byassaying the level of binding that occurs over a period of time. Unboundlabel or detection reagent is then removed and bound label or detectionreagent is detected using a suitable assay or analytical instrument. Themethod employed for detecting the reporter group depends upon the natureof the reporter group. For radioactive labels, scintillation counting orautoradiographic methods are generally appropriate. Spectroscopicmethods may be used to detect dyes, luminescent or chemiluminescentmoieties and various chromogens, fluorescent labels and such like.Biotin may be detected using avidin, coupled to a different reportergroup (commonly a radioactive or fluorescent group or an enzyme). Enzymereporter groups (e.g., horseradish peroxidase, β-galactosidase, alkalinephosphatase and glucose oxidase) may generally be detected by theaddition of substrate (generally for a specific period of time),followed by spectroscopic or other analysis of the reaction products.Regardless of the specific method employed, a level of bound detectionreagent that is at least two fold greater than background (i.e., thelevel observed for a biological sample obtained from a disease-freeindividual) indicates the presence of a malignant disease associatedwith expression of the selected peptide or polypeptide.

In general, methods for monitoring the effectiveness of an immunizationor therapy involve monitoring changes in the level of antibodies or Tcells specific for the selected peptide or polypeptide in a sample, orin an animal such as a human patient. Methods in which antibody levelsare monitored may comprise the steps of: (a) incubating a firstbiological sample, obtained from a patient prior to a therapy orimmunization, with a selected peptide or polypeptide, wherein theincubation is performed under conditions and for a time sufficient toallow immunocomplexes to form; (b) detecting immunocomplexes formedbetween the selected peptide or polypeptide and antibodies or antigenbinding fragments in the biological sample that specifically bind to theselected peptide or polypeptide; (c) repeating steps (a) and (b) using asecond biological sample taken from the patient at later time, such asfor example, following a given therapy or immunization; and (d)comparing the number of immunocomplexes detected in the first and secondbiological samples. Alternatively, a polynucleotide encoding theselected peptide or polypeptide, or an APC expressing the selectedpeptide or polypeptide may be employed in place of the selected peptideor polypeptide itself. Within such methods, immunocomplexes between theselected peptide or polypeptide encoded by a polynucleotide, orexpressed by the APC, and antibodies and/or antigen binding fragments inthe biological sample are detected.

Methods in which T cell activation and/or the number of hematologicalmalignancy polypeptide-specific precursors are monitored may comprisethe steps of: (a) incubating a first biological sample comprising CD4⁺and/or CD8⁺ cells (e.g., bone marrow, peripheral blood or a fractionthereof), obtained from a patient prior to a therapy or immunization,with a hematological malignancy peptide or polypeptide, wherein theincubation is performed under conditions and for a time sufficient toallow specific activation, proliferation and/or lysis of T cells; (b)detecting an amount of activation, proliferation and/or lysis of the Tcells; (c) repeating steps (a) and (b) using a second biological samplecomprising CD4⁺ and/or CD8⁺ T cells, and taken from the same patientfollowing therapy or immunization; and (d) comparing the amount ofactivation, proliferation and/or lysis of T cells in the first andsecond biological samples. Alternatively, a polynucleotide encoding ahematological malignancy related peptide, or an APC expressing such apeptide may be employed in place of the hematological malignancy peptideitself.

A biological sample for use within such methods may be any sampleobtained from a patient that would be expected to contain antibodies,CD4⁺ T cells and/or CD8⁺ T cells. Suitable biological samples includeblood, sera, ascites, bone marrow, pleural effusion and cerebrospinalfluid. A first biological sample may be obtained prior to initiation oftherapy or immunization or part way through a therapy or vaccinationregime. The second biological sample should be obtained in a similarmanner, but at a time following additional therapy or immunization. Thesecond biological sample may be obtained at the completion of, or partway through, therapy or immunization, provided that at least a portionof therapy or immunization takes place between the isolation of thefirst and second biological samples.

Incubation and detection steps for both samples may generally beperformed as described above. A statistically significant increase inthe number of immunocomplexes in the second sample relative to the firstsample reflects successful therapy or immunization.

4.6 Administration of Pharmaceutical Compositions and Formulations

In certain embodiments, the present invention concerns formulation ofone or more of the polynucleotide, polypeptide, peptide, antibody, orantigen binding fragment compositions disclosed herein inpharmaceutically acceptable solutions for administration to a cell or ananimal either alone, or in combination with one or more other modalitiesof anti-cancer therapy, or in combination with one or more diagnostic ortherapeutic agents.

It will also be understood that, if desired, the nucleic acid segment,RNA, or DNA compositions disclosed herein may be administered incombination with other agents as well such as, e.g., proteins orpeptides or various pharmaceutically-active agents. As long as thecomposition comprises at least one of the genetic expression constructsdisclosed herein, there is virtually no limit to other components thatmay also be included, given that the additional agents do not cause asignificant adverse effect upon contact with the target cells or hosttissues. The RNA- or DNA-derived compositions may thus be deliveredalong with various other agents as required in the particular instance.Such RNA or DNA compositions may be purified from host cells or otherbiological sources, or alternatively may be chemically synthesized asdescribed herein. Likewise, such compositions may comprise substitutedor derivatized RNA or DNA compositions. Such compositions may includeone or more therapeutic gene constructs, either alone, or in combinationwith one or more modified peptide or nucleic acid substituentderivatives, and/or other anticancer therapeutics.

The formulation of pharmaceutically-acceptable excipients and carriersolutions are well-known to those of skill in the art, as is thedevelopment of suitable dosing and treatment regimens for using theparticular compositions described herein in a variety of treatmentregimens, including e.g., oral, intravenous, intranasal, transdermal,intraprostatic, intratumoral, and/or intramuscular administration andformulation.

4.6.1 Injectable Delivery

For example, the pharmaceutical compositions disclosed herein may beadministered parenterally, intravenously, intramuscularly, or evenintraperitoneally as described in U.S. Pat. No. 5,543,158, U.S. Pat. No.5,641,515 and U.S. Pat. No. 5,399,363 (each specifically incorporatedherein by reference in its entirety). Solutions of the active compoundsas free-base or pharmacologically acceptable salts may be prepared inwater suitably mixed with a surfactant, such as hydroxypropylcellulose.Dispersions may also be prepared in glycerol, liquid polyethyleneglycols, and mixtures thereof and in oils. Under ordinary conditions ofstorage and use, these preparations contain a preservative to preventthe growth of microorganisms.

The pharmaceutical forms suitable for injectable use include sterileaqueous solutions or dispersions and sterile powders for theextemporaneous preparation of sterile injectable solutions ordispersions (U.S. Pat. No. 5,466,468, specifically incorporated hereinby reference in its entirety). In all cases the form must be sterile andmust be fluid to the extent that easy syringability exists. It must bestable under the conditions of manufacture and storage and must bepreserved against the contaminating action of microorganisms, such asbacteria and fungi. The carrier can be a solvent or dispersion mediumcontaining, for example, water, ethanol, polyol (e.g., glycerol,propylene glycol, and liquid polyethylene glycol, and the like),suitable mixtures thereof, and/or vegetable oils. Proper fluidity may bemaintained, for example, by the use of a coating, such as lecithin, bythe maintenance of the required particle size in the case of dispersionand by the use of surfactants. The prevention of the action ofmicroorganisms can be brought about by various antibacterial andantifungal agents, for example, parabens, chlorobutanol, phenol, sorbicacid, thimerosal, and the like. In many cases, it will be preferable toinclude isotonic agents, for example, sugars or sodium chloride.Prolonged absorption of the injectable compositions can be brought aboutby the use in the compositions of agents delaying absorption, forexample, aluminum monostearate and gelatin.

For parenteral administration in an aqueous solution, for example, thesolution should be suitably buffered if necessary and the liquid diluentfirst rendered isotonic with sufficient saline or glucose. Theseparticular aqueous solutions are especially suitable for intravenous,intramuscular, subcutaneous and intraperitoneal administration. In thisconnection, sterile aqueous media that can be employed will be known tothose of skill in the art in light of the present disclosure. Forexample, one dosage may be dissolved in 1 ml of isotonic NaCl solutionand either added to 1000 ml of hypodermoclysis fluid or injected at theproposed site of infusion, (see for example, Hoover, 1975). Somevariation in dosage will necessarily occur depending on the condition ofthe subject being treated. The person responsible for administrationwill, in any event, determine the appropriate dose for the individualsubject. Moreover, for human administration, preparations should meetsterility, pyrogenicity, and general safety and purity standards asrequired by FDA Office of Biologics standards.

Sterile injectable solutions may be prepared by incorporating the genetherapy constructs in the required amount in the appropriate solventwith several of the other ingredients enumerated above, as required,followed by filtered sterilization. Generally, dispersions are preparedby incorporating the various sterilized active ingredients into asterile vehicle which contains the basic dispersion medium and therequired other ingredients from those enumerated above. In the case ofsterile powders for the preparation of sterile injectable solutions, thepreferred methods of preparation are vacuum-drying and freeze-dryingtechniques which yield a powder of the active ingredient plus anyadditional desired ingredient from a previously sterile-filteredsolution thereof.

The compositions disclosed herein may be formulated in a neutral or saltform Pharmaceutically-acceptable salts, include the acid addition saltsand which are formed with inorganic acids such as, for example,hydrochloric or phosphoric acids, or such organic acids as acetic,oxalic, tartaric, mandelic, and the like. Salts formed with the freecarboxyl groups can also be derived from inorganic bases such as, forexample, sodium, potassium, ammonium, calcium, or ferric hydroxides, andsuch organic bases as isopropylamine, trimethylamine, histidine,procaine and the like. Upon formulation, solutions will be administeredin a manner compatible with the dosage formulation and in such amount asis therapeutically effective. The formulations are easily administeredin a variety of dosage forms such as injectable solutions, drug releasecapsules and the like.

As used herein, “carrier” includes any and all solvents, dispersionmedia, vehicles, coatings, diluents, antibacterial and antifungalagents, isotonic and absorption delaying agents, buffers, carriersolutions, suspensions, colloids, and the like. The use of such mediaand agents for pharmaceutical active substances is well known in theart. Except insofar as any conventional media or agent is incompatiblewith the active ingredient, its use in the therapeutic compositions iscontemplated. Supplementary active ingredients can also be incorporatedinto the compositions.

4.6.2 Intranasal Delivery

One may use nasal solutions or sprays, aerosols or even inhalants forthe treatment of hematological malignancies with one of more of thedisclosed peptides and polynucleotides. Nasal solutions are usuallyaqueous solutions designed for administration to the nasal passages indrops or sprays. Nasal solutions are prepared so that they are similarin many respects to nasal secretions, so that normal ciliary action ismaintained. Thus, the aqueous nasal solutions usually are isotonic andslightly buffered to maintain a pH of from about 5.5 to about 6.5. Inaddition, antimicrobial preservatives, similar to those used inophthalmic preparations, and appropriate drug stabilizers, if required,may be included in the formulation. Various commercial nasalpreparations are known.

Inhalations and inhalants are pharmaceutical preparations designed fordelivering a drug or compound into the respiratory tree of a patient. Avapor or mist is administered and reaches the affected area, often togive relief from symptoms of bronchial and nasal congestion. However,this route can also be employed to deliver agents into the systemiccirculation. Inhalations may be administered by the nasal or oralrespiratory routes. The administration of inhalation solutions is onlyeffective if the droplets are sufficiently fine and uniform in size sothat the mist reaches the bronchioles.

Another group of products, also known as inhalations, and sometimescalled insufflations, consists of finely powdered or liquid drugs thatare carried into the respiratory passages by the use of special deliverysystems, such as pharmaceutical aerosols, that hold a solution orsuspension of the drug in a liquefied gas propellant. When releasedthrough a suitable valve and oral adapter, a metered does of theinhalation is propelled into the respiratory tract of the patient.

Particle size is of importance in the administration of this type ofpreparation. It has been reported that the optimum particle size forpenetration into the pulmonary cavity is of the order of about 0.5 toabout 7 μm. Fine mists are produced by pressurized aerosols and hencetheir use in considered advantageous.

4.6.3 Liposome-, Nanocapsule-, and Microparticle-Mediated Delivery

In certain embodiments, the inventors contemplate the use of liposomes,nanocapsules, microparticles, microspheres, lipid particles, vesicles,and the like, for the introduction of the polynucleotide compositions ofthe present invention into suitable host cells. In particular, thepolynucleotide compositions of the present invention may be formulatedfor delivery either encapsulated in a lipid particle, a liposome, avesicle, a nanosphere, or a nanoparticle or the like.

Such formulations may be preferred for the introduction ofpharmaceutically acceptable formulations of the nucleic acids disclosedherein. The formation and use of liposomes is generally known to thoseof skill in the art (see for example, Couvreur et al., 1977; Couvreur,1988; Lasic, 1998; which describes the use of liposomes and nanocapsulesin the targeted antibiotic therapy for intracellular bacterialinfections and diseases). Recently, liposomes were developed withimproved serum stability and circulation half-lives (Gabizon andPapahadjopoulos, 1988; Allen and Choun, 1987; U.S. Pat. No. 5,741,516,specifically incorporated herein by reference in its entirety). Further,various methods of liposome and liposome like preparations as potentialdrug carriers have been reviewed (Takakura, 1998; Chandran et al., 1997;Margalit, 1995; U.S. Pat. No. 5,567,434; U.S. Pat. No. 5,552,157; U.S.Pat. No. 5,565,213; U.S. Pat. No. 5,738,868 and U.S. Pat. No. 5,795,587,each specifically incorporated herein by reference in its entirety).

Liposomes have been used successfully with a number of cell types thatare normally resistant to transfection by other procedures including Tcell suspensions, primary hepatocyte cultures and PC12 cells (Renneisenet al., 1990; Muller et al., 1990). In addition, liposomes are free ofthe DNA length constraints that are typical of viral-based deliverysystems. Liposomes have been used effectively to introduce genes, drugs(Heath and Martin, 1986; Heath et al., 1986; Balazsovits et al., 1989;Fresta and Puglisi, 1996), radiotherapeutic agents (Pikul et al., 1987),enzymes (Imaizumi et al., 1990a; Imaizumi et al., 1990b), viruses(Faller and Baltimore, 1984), transcription factors and allostericeffectors (Nicolau and Gersonde, 1979) into a variety of cultured celllines and animals. In addition, several successful clinical trailsexamining the effectiveness of liposome-mediated drug delivery have beencompleted (Lopez-Berestein et al., 1985a; 1985b; Coune, 1988; Sculier etal., 1988). Furthermore, several studies suggest that the use ofliposomes is not associated with autoimmune responses, toxicity orgonadal localization after systemic delivery (Mori and Fukatsu, 1992).

Liposomes are formed from phospholipids that are dispersed in an aqueousmedium and spontaneously form multilamellar concentric bilayer vesicles(also termed multilamellar vesicles (MLVs). MLVs generally havediameters of from 25 nm to 4 μm. Sonication of MLVs results in theformation of small unilamellar vesicles (SUVs) with diameters in therange of 200 to 500 Å, containing an aqueous solution in the core.

Liposomes bear resemblance to cellular membranes and are contemplatedfor use in connection with the present invention as carriers for thepeptide compositions. They are widely suitable as both water- andlipid-soluble substances can be entrapped, i.e. in the aqueous spacesand within the bilayer itself, respectively. It is possible that thedrug-bearing liposomes may even be employed for site-specific deliveryof active agents by selectively modifying the liposomal formulation.

In addition to the teachings of Couvreur et al. (1977; 1988), thefollowing information may be utilized in generating liposomalformulations. Phospholipids can form a variety of structures other thanliposomes when dispersed in water, depending on the molar ratio of lipidto water. At low ratios the liposome is the preferred structure. Thephysical characteristics of liposomes depend on pH, ionic strength andthe presence of divalent cations. Liposomes can show low permeability toionic and polar substances, but at elevated temperatures undergo a phasetransition which markedly alters their permeability. The phasetransition involves a change from a closely packed, ordered structure,known as the gel state, to a loosely packed, less-ordered structure,known as the fluid state. This occurs at a characteristicphase-transition temperature and results in an increase in permeabilityto ions, sugars, and drugs.

Alternatively, the invention provides for pharmaceutically acceptablenanocapsule formulations of the polynucleotide compositions of thepresent invention. Nanocapsules can generally entrap compounds in astable and reproducible way (Henry-Michelland et al., 1987;Quintanar-Guerrero et al., 1998; Douglas et al., 1987). To avoid sideeffects due to intracellular polymeric overloading, such ultrafineparticles (sized around 0.1 μm) should be designed using polymers ableto be degraded in vivo. Biodegradable polyalkyl-cyanoacrylatenanoparticles that meet these requirements are contemplated for use inthe present invention, and such particles may be are easily made, asdescribed (Couvreur et al., 1980; 1988; zur Muhlen et al., 1998; Zambauxet al. 1998; Pinto-Alphandry et al., 1995 and U.S. Pat. No. 5,145,684,specifically incorporated herein by reference in its entirety). Inparticular, methods of polynucleotide delivery to a target cell usingeither nanoparticles or nanospheres (Schwab et al., 1994; Truong-Le etal., 1998) are also particularly contemplated to be useful informulating the disclosed compositions for administration to an animal,and to a human in particular.

4.7 Therapeutic Agents and Kits

The invention also provides one or more of the hematologicalmalignancy-related compositions formulated with one or morepharmaceutically acceptable excipients, carriers, diluents, adjuvants,and/or other components for use in the preparation of medicaments, ordiagnostic reagents, as well as various kits comprising one or more ofsuch compositions, medicaments, or formulations intended foradministration to an animal in need thereof, or for use in one or morediagnostic assays for identifying polynucleotides, polypeptides, and/orantibodies that are specific for one or more hematologicalmalignancy-related compounds as described herein. In addition to thedisclosed epitopes, antibodies and antigen binding fragments, antibody-or antigen binding fragment-encoding polynucleotides or additionalanticancer agents, polynucleotides, peptides, antigens, or othertherapeutic compounds as may be employed in the formulation ofparticular compositions and formulations disclosed herein, andparticularly in the preparation of anticancer agents oranti-hematological malignancies therapies for administration to theaffected mammal.

As such, preferred animals for administration of the pharmaceuticalcompositions disclosed herein include mammals, and particularly humans.Other preferred animals include primates, sheep, goats, bovines,equines, porcines, lupines, canines, and felines, as well as any othermammalian species commonly considered pets, livestock, or commerciallyrelevant animal species. The compositions and formulations may includepartially or significantly purified polypeptide, polynucleotide, orantibody or antigen binding fragment compositions, either alone, or incombination with one or more additional active ingredients, anticanceragents, vaccines, adjuvants, or other therapeutics which may be obtainedfrom natural or recombinant sources, or which may be obtainablenaturally or either chemically synthesized, or alternatively produced invitro from recombinant host cells expressing one or more nucleic acidsegments that encode one or more such additional active ingredients,carriers, adjuvants, cofactors, or other therapeutic compound.

4.8 Diagnostic Reagents and Kits

The invention further provides diagnostic reagents and kits comprisingone or more such reagents for use in a variety of diagnostic assays,including for example, immunoassays such as ELISA and “sandwich”-typeimmunoassays. Such kits may preferably include at least a first peptide,or a first antibody or antigen binding fragment of the invention, afunctional fragment thereof, or a cocktail thereof, and means for signalgeneration. The kit's components may be pre-attached to a solid support,or may be applied to the surface of a solid support when the kit isused. The signal generating means may come pre-associated with anantibody of the invention or may require combination with one or morecomponents, e.g., buffers, antibody-enzyme conjugates, enzymesubstrates, or the like, prior to use. Kits may also include additionalreagents, e.g., blocking reagents for reducing nonspecific binding tothe solid phase surface, washing reagents, enzyme substrates, and thelike. The solid phase surface may be in the form of microtiter plates,microspheres, or other materials suitable for immobilizing proteins,peptides, or polypeptides. Preferably, an enzyme that catalyzes theformation of a chemiluminescent or chromogenic product or the reductionof a chemiluminescent or chromogenic substrate is a component of thesignal generating means. Such enzymes are well known in the art.

Such kits are useful in the detection, monitoring and diagnosis ofconditions characterized by over-expression or inappropriate expressionof hematological malignancy-related peptides, polypeptides, antibodies,and/or polynucleotides, as well as hybridomas, host cells, and vectorscomprising one or more such compositions as disclosed herein.

The therapeutic and diagnostic kits of the present invention may also beprepared that comprise at least one of the antibody, peptide, antigenbinding fragment, hybridoma, vector, vaccine, polynucleotide, orcellular compositions disclosed herein and instructions for using thecomposition as a diagnostic reagent or therapeutic agent. Containers foruse in such kits may typically comprise at least one vial, test tube,flask, bottle, syringe or other suitable container, into which one ormore of the diagnostic and/or therapeutic composition(s) may be placed,and preferably suitably aliquoted. Where a second therapeutic agent isalso provided, the kit may also contain a second distinct container intowhich this second diagnostic and/or therapeutic composition may beplaced. Alternatively, a plurality of compounds may be prepared in asingle pharmaceutical composition, and may be packaged in a singlecontainer means, such as a vial, flask, syringe, bottle, or othersuitable single container. The kits of the present invention will alsotypically include a means for containing the vial(s) in closeconfinement for commercial sale, such as, e.g., injection or blow-moldedplastic containers into which the desired vial(s) are retained. Where aradiolabel, chromogenic, fluorigenic, or other type of detectable labelor detecting means is included within the kit, the labeling agent may beprovided either in the same container as the diagnostic or therapeuticcomposition itself, or may alternatively be placed in a second distinctcontainer means into which this second composition may be placed andsuitably aliquoted. Alternatively, the detection reagent and the labelmay be prepared in a single container means, and in most cases, the kitwill also typically include a means for containing the vial(s) in closeconfinement for commercial sale and/or convenient packaging anddelivery.

4.9 Polynucleotide Compositions

As used herein, the terms “DNA segment” and “polynucleotide” refer to aDNA molecule that has been isolated free of total genomic DNA of aparticular species. Therefore, a DNA segment encoding a polypeptiderefers to a DNA segment that contains one or more coding sequences yetis substantially isolated away from, or purified free from, totalgenomic DNA of the species from which the DNA segment is obtained.Included within the terms “DNA segment” and “polynucleotide” are DNAsegments and smaller fragments of such segments, and also recombinantvectors, including, for example, plasmids, cosmids, phagemids, phage,viruses, and the like.

As will be understood by those skilled in the art, the DNA segments ofthis invention can include genomic sequences, extra-genomic andplasmid-encoded sequences and smaller engineered gene segments thatexpress, or may be adapted to express, proteins, polypeptides, peptidesand the like. Such segments may be naturally isolated, or modifiedsynthetically by the hand of man.

“Isolated,” as used herein, means that a polynucleotide is substantiallyaway from other coding sequences, and that the DNA segment does notcontain large portions of unrelated coding DNA, such as largechromosomal fragments or other functional genes or polypeptide codingregions. Of course, this refers to the DNA segment as originallyisolated, and does not exclude genes or coding regions later added tothe segment by the hand of man.

As will be recognized by the skilled artisan, polynucleotides may besingle-stranded (coding or antisense) or double-stranded, and may be DNA(genomic, cDNA or synthetic) or RNA molecules. RNA molecules includeHnRNA molecules, which contain introns and correspond to a DNA moleculein a one-to-one manner, and mRNA molecules, which do not containintrons. Additional coding or non-coding sequences may, but need not, bepresent within a polynucleotide of the present invention, and apolynucleotide may, but need not, be linked to other molecules and/orsupport materials.

Polynucleotides may comprise a native sequence (i.e., an endogenoussequence that encodes a hematological malignancy-related tumor proteinor a portion thereof) or may comprise a variant, or a biological orantigenic functional equivalent of such a sequence. Polynucleotidevariants may contain one or more substitutions, additions, deletionsand/or insertions, as further described below, preferably such that theimmunogenicity of the encoded polypeptide is not diminished, relative toa native tumor protein. The effect on the immunogenicity of the encodedpolypeptide may generally be assessed as described herein. The term“variants” also encompasses homologous genes of xenogenic origin.

When comparing polynucleotide or polypeptide sequences, two sequencesare said to be “identical” if the sequence of nucleotides or amino acidsin the two sequences is the same when aligned for maximumcorrespondence, as described below. Comparisons between two sequencesare typically performed by comparing the sequences over a comparisonwindow to identify and compare local regions of sequence similarity. A“comparison window” as used herein, refers to a segment of at leastabout 20 contiguous positions, usually 30 to about 75, 40 to about 50,in which a sequence may be compared to a reference sequence of the samenumber of contiguous positions after the two sequences are optimallyaligned.

Optimal alignment of sequences for comparison may be conducted using theMegalign program in the Lasergene suite of bioinformatics software(DNASTAR, Inc., Madison, Wis.), using default parameters. This programembodies several alignment schemes described in the followingreferences: Dayhoff, M. O. (1978) A model of evolutionary change inproteins—Matrices for detecting distant relationships. In Dayhoff, M. O.(ed.) Atlas of Protein Sequence and Structure, National BiomedicalResearch Foundation, Washington D.C. Vol. 5, Suppl. 3, pp. 345-358; HeinJ. (1990) Unified Approach to Alignment and Phylogenes pp. 626-645Methods in Enzymology vol. 183, Academic Press, Inc., San Diego, Calif.;Higgins, D. G. and Sharp, P. M. (1989) CABIOS 5:151-153; Myers, E. W.and Muller W. (1988) CABIOS 4:11-17; Robinson, E. D. (1971) Comb. Theor11:105; Santou, N. Nes, M. (1987) Mol. Biol. Evol. 4:406-425; Sneath, P.H. A. and Sokal, R. R. (1973) Numerical Taxonomy—the Principles andPractice of Numerical Taxonomy, Freeman Press, San Francisco, Calif.;Wilbur, W. J. and Lipman, D. J. (1983) Proc. Natl. Acad., Sci. USA80:726-730.

Alternatively, optimal alignment of sequences for comparison may beconducted by the local identity algorithm of Smith and Waterman (1981)Add. APL. Math 2:482, by the identity alignment algorithm of Needlemanand Wunsch (1970) J. Mol. Biol. 48:443, by the search for similaritymethods of Pearson and Lipman (1988) Proc. Natl. Acad. Sci. USA 85:2444, by computerized implementations of these algorithms (GAP, BESTFIT,BLAST, FASTA, and TFASTA in the Wisconsin Genetics Software Package,Genetics Computer Group (GCG), 575 Science Dr., Madison, Wis.), or byinspection.

One preferred example of algorithms that are suitable for determiningpercent sequence identity and sequence similarity are the BLAST andBLAST 2.0 algorithms, which are described in Altschul et al. (1977)Nucl. Acids Res. 25:3389-3402 and Altschul et al. (1990) J. Mol. Biol.215:403-410, respectively. BLAST and BLAST 2.0 can be used, for examplewith the parameters described herein, to determine percent sequenceidentity for the polynucleotides and polypeptides of the invention.Software for performing BLAST analyses is publicly available through theNational Center for Biotechnology Information. In one illustrativeexample, cumulative scores can be calculated using, for nucleotidesequences, the parameters M (reward score for a pair of matchingresidues; always >0) and N (penalty score for mismatching residues;always <0). For amino acid sequences, a scoring matrix can be used tocalculate the cumulative score. Extension of the word hits in eachdirection are halted when: the cumulative alignment score falls off bythe quantity X from its maximum achieved value; the cumulative scoregoes to zero or below, due to the accumulation of one or morenegative-scoring residue alignments; or the end of either sequence isreached. The BLAST algorithm parameters W, T and X determine thesensitivity and speed of the alignment. The BLASTN program (fornucleotide sequences) uses as defaults a wordlength (W) of 11, andexpectation (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff andHenikoff (1989) Proc. Natl. Acad. Sci. USA 89:10915) alignments, (B) of50, expectation (E) of 10, M=5, N=−4 and a comparison of both strands.

Preferably, the “percentage of sequence identity” is determined bycomparing two optimally aligned sequences over a window of comparison ofat least 20 positions, wherein the portion of the polynucleotide orpolypeptide sequence in the comparison window may comprise additions ordeletions (i.e., gaps) of 20 percent or less, usually 5 to 15 percent,or 10 to 12 percent, as compared to the reference sequences (which doesnot comprise additions or deletions) for optimal alignment of the twosequences. The percentage is calculated by determining the number ofpositions at which the identical nucleic acid bases or amino acidresidue occurs in both sequences to yield the number of matchedpositions, dividing the number of matched positions by the total numberof positions in the reference sequence (i.e., the window size) andmultiplying the results by 100 to yield the percentage of sequenceidentity.

Therefore, the present invention encompasses polynucleotide andpolypeptide sequences having substantial identity to the sequencesdisclosed herein, for example those comprising at least 50% sequenceidentity, preferably at least 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%,95%, 96%, 97%, 98%, or 99% or higher, sequence identity compared to apolynucleotide or polypeptide sequence of this invention using themethods described herein, (e.g. BLAST analysis using standardparameters, as described below). One skilled in this art will recognizethat these values can be appropriately adjusted to determinecorresponding identity of proteins encoded by two nucleotide sequencesby taking into account codon degeneracy, amino acid similarity, readingframe positioning and the like.

In additional embodiments, the present invention provides isolatedpolynucleotides and polypeptides comprising various lengths ofcontiguous stretches of sequence identical to or complementary to one ormore of the sequences disclosed herein. For example, polynucleotides areprovided by this invention that comprise at least about 15, 20, 30, 40,50, 75, 100, 150, 200, 300, 400, 500 or 1000 or more contiguousnucleotides of one or more of the sequences disclosed herein as well asall intermediate lengths there between. It will be readily understoodthat “intermediate lengths”, in this context, means any length betweenthe quoted values, such as 16, 17, 18, 19, etc.; 21, 22, 23, etc.; 30,31, 32, etc.; 50, 51, 52, 53, etc.; 100, 101, 102, 103, etc.; 150, 151,152, 153, etc.; including all integers through 200-500; 500-1,000, andthe like.

The polynucleotides of the present invention, or fragments thereof,regardless of the length of the coding sequence itself, may be combinedwith other DNA sequences, such as promoters, polyadenylation signals,additional restriction enzyme sites, multiple cloning sites, othercoding segments, and the like, such that their overall length may varyconsiderably. It is therefore contemplated that a nucleic acid fragmentof almost any length may be employed, with the total length preferablybeing limited by the ease of preparation and use in the intendedrecombinant DNA protocol. For example, illustrative DNA segments withtotal lengths of about 10,000, about 5000, about 3000, about 2,000,about 1,000, about 500, about 200, about 100, about 50 base pairs inlength, and the like, (including all intermediate lengths) arecontemplated to be useful in many implementations of this invention.

In other embodiments, the present invention is directed topolynucleotides that are capable of hybridizing under moderatelystringent conditions to a polynucleotide sequence provided herein, or afragment thereof, or a complementary sequence thereof. Hybridizationtechniques are well known in the art of molecular biology. For purposesof illustration, suitable moderately stringent conditions for testingthe hybridization of a polynucleotide of this invention with otherpolynucleotides include prewashing in a solution of 5×SSC, 0.5% SDS, 1.0mM EDTA (pH 8.0); hybridizing at 50° C.-65° C., 5×SSC, overnight;followed by washing twice at 65° C. for 20 minutes with each of 2×, 0.5×and 0.2×SSC containing 0.1% SDS.

Moreover, it will be appreciated by those of ordinary skill in the artthat, as a result of the degeneracy of the genetic code, there are manynucleotide sequences that encode a polypeptide as described herein. Someof these polynucleotides bear minimal homology to the nucleotidesequence of any native gene. Nonetheless, polynucleotides that vary dueto differences in codon usage are specifically contemplated by thepresent invention. Further, alleles of the genes comprising thepolynucleotide sequences provided herein are within the scope of thepresent invention. Alleles are endogenous genes that are altered as aresult of one or more mutations, such as deletions, additions and/orsubstitutions of nucleotides. The resulting mRNA and protein may, butneed not, have an altered structure or function. Alleles may beidentified using standard techniques (such as hybridization,amplification and/or database sequence comparison).

4.10 Probes and Primers

In other embodiments of the present invention, the polynucleotidesequences provided herein can be advantageously used as probes orprimers for nucleic acid hybridization. As such, it is contemplated thatnucleic acid segments that comprise a sequence region of at least about15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, or 95nucleotide long contiguous sequence that has the same sequence as, or iscomplementary to, at least a 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65,70, 75, 80, 85, 90, or 95 nucleotide long contiguous sequence thedisclosed polynucleotides will find particular utility in a variety ofhybridization embodiments. Longer contiguous identical or complementarysequences, e.g., those of about 100, 110, 120, 130, 140, 150, 160, 170,180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310,320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450,460, 470, 480, 490, 500, 525, 550, 575, 600, 650, 700, 750, 800, 850,900, 950, or even 1000 or so nucleotides (including all intermediatelengths) and all full-length sequences as the disclosed polynucleotideswill also be of use in certain embodiments as probes, primers, oramplification targets and such like.

The ability of such nucleic acid probes to specifically hybridize to asequence of interest will enable them to be of use in detecting thepresence of complementary sequences in a given sample. However, otheruses are also envisioned, such as the use of the sequence informationfor the preparation of mutant species primers, or primers, for use inpreparing other genetic constructions, and for identifying andcharacterizing full-length polynucleotides and full, or substantiallyfull-length cDNAs, mRNAs, and such like.

Polynucleotide molecules having sequence regions consisting ofcontiguous nucleotide stretches identical or complementary to one ormore polynucleotide sequences as disclosed herein, are particularlycontemplated as hybridization probes for use in, e.g. Southernhybridization analyses and Northern blotting. This would allow a geneproduct, or fragment thereof, to be analyzed, both in diverse cell typesand also in various bacterial cells. The total size of fragment, as wellas the size of the complementary stretch(es), will ultimately depend onthe intended use or application of the particular nucleic acid segment.Smaller fragments will generally find use in hybridization embodiments,wherein the length of the contiguous complementary region may be varied,such as between about 15, 20, 25, 30, 35, 40, 45, 50, 55, 60 or so andup to and including larger contiguous complementary sequences, includingthose of about 70, 80, 90, 100, 120, 140, 160, 180, or 200 or sonucleotides in length may also be used, according to the given desiredgoal, and the particular length of the complementary sequences onewishes to detect by hybridization analysis.

The use of a hybridization probe of about between about 20 and about 500nucleotides in length allows the formation of a duplex molecule that isboth stable and selective. Molecules having contiguous complementarysequences over stretches greater than about 20 or so bases in length aregenerally preferred, though, in order to increase stability andselectivity of the hybrid, and thereby improve the quality and degree ofspecific hybrid molecules obtained. One will generally prefer to designnucleic acid molecules having gene-complementary stretches of betweenabout 25 and 300 or so contiguous nucleotides, or even longer wheredesired.

Hybridization probes may be selected from any portion of any of thesequences disclosed herein. All that is required is to review thedisclosed sequences, or to any contiguous portion of such a sequence,from about 15 to 30 nucleotides in length up to and including the fulllength sequence, that one wishes to utilize as a probe or primer. Thechoice of probe and primer sequences may be governed by various factors.For example, one may wish to employ primers from towards the termini ofthe total sequence.

Small polynucleotide segments or fragments may be readily prepared by,for example, directly synthesizing the fragment by chemical means, as iscommonly practiced using an automated oligonucleotide synthesizer. Also,fragments may be obtained by application of nucleic acid reproductiontechnology, such as the PCR™ technology of U.S. Pat. No. 4,683,202(incorporated herein by reference), by introducing selected sequencesinto recombinant vectors for recombinant production, and by otherrecombinant DNA techniques generally known to those of skill in the artof molecular biology.

The nucleotide sequences of the invention may be used for their abilityto selectively form duplex molecules with complementary stretches of theentire gene or gene fragments of interest. Depending on the applicationenvisioned, one will typically desire to employ varying conditions ofhybridization to achieve varying degrees of selectivity of probe towardstarget sequence. For applications requiring high selectivity, one willtypically desire to employ relatively stringent conditions to form thehybrids, e.g., one will select relatively low salt and/or hightemperature conditions, such as provided by a salt concentration of fromabout 0.02 M to about 0.15 M salt at temperatures of from about 50° C.to about 70° C. Such selective conditions tolerate little, if any,mismatch between the probe and the template or target strand, and wouldbe particularly suitable for isolating related sequences.

Of course, for some applications, for example, where one desires toprepare mutants employing a mutant primer strand hybridized to anunderlying template, less stringent (reduced stringency) hybridizationconditions will typically be needed in order to allow formation of theheteroduplex. In these circumstances, one may desire to employ saltconditions such as those of from about 0.15 M to about 0.9 M salt, attemperatures ranging from about 20° C. to about 55° C. Cross-hybridizingspecies can thereby be readily identified as positively hybridizingsignals with respect to control hybridizations. In any case, it isgenerally appreciated that conditions can be rendered more stringent bythe addition of increasing amounts of formamide, which serves todestabilize the hybrid duplex in the same manner as increasedtemperature. Thus, hybridization conditions can be readily manipulated,and thus will generally be a method of choice depending on the desiredresults.

4.11 Polynucleotide Identification and Characterization

Polynucleotides may be identified, prepared and/or manipulated using anyof a variety of well established techniques. For example, apolynucleotide may be identified, as described in more detail below, byscreening a microarray of cDNAs for tumor-associated expression (i.e.,expression that is at least two fold greater in a tumor than in normaltissue, as determined using a representative assay provided herein).Such screens may be performed, for example, using a Synteni microarray(Palo Alto, Calif.) according to the manufacturer's instructions (andessentially as described by Schena et al., Proc. Natl. Acad. Sci. USA93:10614-10619, 1996 and Heller et al., Proc. Natl. Acad. Sci. USA94:2150-2155, 1997). Alternatively, polynucleotides may be amplifiedfrom cDNA prepared from cells expressing the proteins described herein,such as hematological malignancy-related tumor cells. Suchpolynucleotides may be amplified via polymerase chain reaction (PCR).For this approach, sequence-specific primers may be designed based onthe sequences provided herein, and may be purchased or synthesized.

An amplified portion of a polynucleotide of the present invention may beused to isolate a full length gene from a suitable library (e.g., ahematological malignancy-related tumor cDNA library) using well knowntechniques. Within such techniques, a library (cDNA or genomic) isscreened using one or more polynucleotide probes or primers suitable foramplification. Preferably, a library is size-selected to include largermolecules. Random primed libraries may also be preferred for identifying5′ and upstream regions of genes. Genomic libraries are preferred forobtaining introns and extending 5′ sequences.

For hybridization techniques, a partial sequence may be labeled (e.g.,by nick-translation or end-labeling with ³²P) using well knowntechniques. A bacterial or bacteriophage library is then generallyscreened by hybridizing filters containing denatured bacterial colonies(or lawns containing phage plaques) with the labeled probe (see Sambrooket al., Molecular Cloning: A Laboratory Manual, Cold Spring HarborLaboratories, Cold Spring Harbor, N.Y., 1989). Hybridizing colonies orplaques are selected and expanded, and the DNA is isolated for furtheranalysis. cDNA clones may be analyzed to determine the amount ofadditional sequence by, for example, PCR using a primer from the partialsequence and a primer from the vector. Restriction maps and partialsequences may be generated to identify one or more overlapping clones.The complete sequence may then be determined using standard techniques,which may involve generating a series of deletion clones. The resultingoverlapping sequences can then assembled into a single contiguoussequence. A full length cDNA molecule can be generated by ligatingsuitable fragments, using well known techniques.

Alternatively, there are numerous amplification techniques for obtaininga full length coding sequence from a partial cDNA sequence. Within suchtechniques, amplification is generally performed via PCR. Any of avariety of commercially available kits may be used to perform theamplification step. Primers may be designed using, for example, softwareor algorithms or formulas well known in the art.

One such amplification technique is inverse PCR (see Triglia et al.,Nucl. Acids Res. 16:8186, 1988), which uses restriction enzymes togenerate a fragment in the known region of the gene. The fragment isthen circularized by intramolecular ligation and used as a template forPCR with divergent primers derived from the known region. Within analternative approach, sequences adjacent to a partial sequence may beretrieved by amplification with a primer to a linker sequence and aprimer specific to a known region. The amplified sequences are typicallysubjected to a second round of amplification with the same linker primerand a second primer specific to the known region. A variation on thisprocedure, which employs two primers that initiate extension in oppositedirections from the known sequence, is described in WO 96/38591. Anothersuch technique is known as “rapid amplification of cDNA ends” or RACE.This technique involves the use of an internal primer and an externalprimer, which hybridizes to a polyA region or vector sequence, toidentify sequences that are 5′ and 3′ of a known sequence. Additionaltechniques include capture PCR (Lagerstrom et al., PCR Methods Applic.1:111-19, 1991) and walking PCR (Parker et al., Nucl. Acids. Res.19:3055-60, 1991). Other methods employing amplification may also beemployed to obtain a full length cDNA sequence.

In certain instances, it is possible to obtain a full length cDNAsequence by analysis of sequences provided in an expressed sequence tag(EST) database, such as that available from GenBank. Searches foroverlapping ESTs may generally be performed using well known programs(e.g., NCBI BLAST searches), and such ESTs may be used to generate acontiguous full length sequence. Full length DNA sequences may also beobtained by analysis of genomic fragments.

4.12 Polynucleotide Expression in Host Cells

In other embodiments of the invention, polynucleotide sequences orfragments thereof which encode polypeptides of the invention, or fusionproteins or functional equivalents thereof, may be used in recombinantDNA molecules to direct expression of a polypeptide in appropriate hostcells. Due to the inherent degeneracy of the genetic code, other DNAsequences that encode substantially the same or a functionallyequivalent amino acid sequence may be produced and these sequences maybe used to clone and express a given polypeptide.

As will be understood by those of skill in the art, it may beadvantageous in some instances to produce polypeptide-encodingnucleotide sequences possessing non-naturally occurring codons. Forexample, codons preferred by a particular prokaryotic or eukaryotic hostcan be selected to increase the rate of protein expression or to producea recombinant RNA transcript having desirable properties, such as ahalf-life which is longer than that of a transcript generated from thenaturally occurring sequence.

Moreover, the polynucleotide sequences of the present invention can beengineered using methods generally known in the art in order to alterpolypeptide encoding sequences for a variety of reasons, including butnot limited to, alterations which modify the cloning, processing, and/orexpression of the gene product. For example, DNA shuffling by randomfragmentation and PCR reassembly of gene fragments and syntheticoligonucleotides may be used to engineer the nucleotide sequences. Inaddition, site-directed mutagenesis may be used to insert newrestriction sites, alter glycosylation patterns, change codonpreference, produce splice variants, or introduce mutations, and soforth.

In another embodiment of the invention, natural, modified, orrecombinant nucleic acid sequences may be ligated to a heterologoussequence to encode a fusion protein. For example, to screen peptidelibraries for inhibitors of polypeptide activity, it may be useful toencode a chimeric protein that can be recognized by a commerciallyavailable antibody. A fusion protein may also be engineered to contain acleavage site located between the polypeptide-encoding sequence and theheterologous protein sequence, so that the polypeptide may be cleavedand purified away from the heterologous moiety.

Sequences encoding a desired polypeptide may be synthesized, in whole orin part, using chemical methods well known in the art (see Caruthers, M.H. et al. (1980) Nucl. Acids Res. Symp. Ser. 215-223, Horn, T. et al.(1980) Nucl. Acids Res. Symp. Ser. 225-232). Alternatively, the proteinitself may be produced using chemical methods to synthesize the aminoacid sequence of a polypeptide, or a portion thereof. For example,peptide synthesis can be performed using various solid-phase techniques(Roberge, J. Y. et al. (1995) Science 269:202-204) and automatedsynthesis may be achieved, for example, using the ABI 431A PeptideSynthesizer (Perkin Elmer, Palo Alto, Calif.).

A newly synthesized peptide may be substantially purified by preparativehigh performance liquid chromatography (e.g., Creighton, T. (1983)Proteins, Structures and Molecular Principles, WH Freeman and Co., NewYork, N.Y.) or other comparable techniques available in the art. Thecomposition of the synthetic peptides may be confirmed by amino acidanalysis or sequencing (e.g., the Edman degradation procedure).Additionally, the amino acid sequence of a polypeptide, or any partthereof, may be altered during direct synthesis and/or combined usingchemical methods with sequences from other proteins, or any partthereof, to produce a variant polypeptide.

In order to express a desired polypeptide, the nucleotide sequencesencoding the polypeptide, or functional equivalents, may be insertedinto appropriate expression vector, i.e., a vector which contains thenecessary elements for the transcription and translation of the insertedcoding sequence. Methods which are well known to those skilled in theart may be used to construct expression vectors containing sequencesencoding a polypeptide of interest and appropriate transcriptional andtranslational control elements. These methods include in vitrorecombinant DNA techniques, synthetic techniques, and in vivo geneticrecombination. Such techniques are described in Sambrook, J. et al.(1989) Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Press,Plainview, N.Y., and Ausubel, F. M. et al. (1989) Current Protocols inMolecular Biology, John Wiley & Sons, New York. N.Y.

A variety of expression vector/host systems may be utilized to containand express polynucleotide sequences. These include, but are not limitedto, microorganisms such as bacteria transformed with recombinantbacteriophage, plasmid, or cosmid DNA expression vectors; yeasttransformed with yeast expression vectors; insect cell systems infectedwith virus expression vectors (e.g., baculovirus); plant cell systemstransformed with virus expression vectors (e.g., cauliflower mosaicvirus, CaMV; tobacco mosaic virus, TMV) or with bacterial expressionvectors (e.g., Ti or pBR322 plasmids); or animal cell systems.

The “control elements” or “regulatory sequences” present in anexpression vector are those non-translated regions of thevector—enhancers, promoters, 5′ and 3′ untranslated regions—whichinteract with host cellular proteins to carry out transcription andtranslation. Such elements may vary in their strength and specificity.Depending on the vector system and host utilized, any number of suitabletranscription and translation elements, including constitutive andinducible promoters, may be used. For example, when cloning in bacterialsystems, inducible promoters such as the hybrid lacZ promoter of thePBLUESCRIPT phagemid (Stratagene, La Jolla, Calif.) or PSPORT1 plasmid(Gibco BRL, Gaithersburg, Md.) and the like may be used. In mammaliancell systems, promoters from mammalian genes or from mammalian virusesare generally preferred. If it is necessary to generate a cell line thatcontains multiple copies of the sequence encoding a polypeptide, vectorsbased on SV40 or EBV may be advantageously used with an appropriateselectable marker.

In bacterial systems, a number of expression vectors may be selecteddepending upon the use intended for the expressed polypeptide. Forexample, when large quantities are needed, for example for the inductionof antibodies, vectors which direct high level expression of fusionproteins that are readily purified may be used. Such vectors include,but are not limited to, the multifunctional E. coli cloning andexpression vectors such as BLUESCRIPT (Stratagene), in which thesequence encoding the polypeptide of interest may be ligated into thevector in frame with sequences for the amino-terminal Met and thesubsequent 7 residues of .beta.-galactosidase so that a hybrid proteinis produced; pIN vectors (Van Heeke, G. and S. M. Schuster (1989) J.Biol. Chem. 264:5503-5509); and the like. pGEX Vectors (Promega,Madison, Wis.) may also be used to express foreign polypeptides asfusion proteins with glutathione S-transferase (GST). In general, suchfusion proteins are soluble and can easily be purified from lysed cellsby adsorption to glutathione-agarose beads followed by elution in thepresence of free glutathione. Proteins made in such systems may bedesigned to include heparin, thrombin, or factor XA protease cleavagesites so that the cloned polypeptide of interest can be released fromthe GST moiety at will.

In the yeast, Saccharomyces cerevisiae, a number of vectors containingconstitutive or inducible promoters such as alpha factor, alcoholoxidase, and PGH may be used. For reviews, see Ausubel et al. (supra)and Grant et al. (1987) Methods Enzymol. 153:516-544.

In cases where plant expression vectors are used, the expression ofsequences encoding polypeptides may be driven by any of a number ofpromoters. For example, viral promoters such as the 35S and 19Spromoters of CaMV may be used alone or in combination with the omegaleader sequence from TMV (Takamatsu, N. (1987) EMBO J. 6:307-311.Alternatively, plant promoters such as the small subunit of RUBISCO orheat shock promoters may be used (Coruzzi, G. et al. (1984) EMBO J.3:1671-1680; Broglie, R. et al. (1984) Science 224:838-843; and Winter,J. et al. (1991) Results Probl. Cell Differ. 17:85-105). Theseconstructs can be introduced into plant cells by direct DNAtransformation or pathogen-mediated transfection. Such techniques aredescribed in a number of generally available reviews (see, for example,Hobbs, S. or Murry, L. E. in McGraw Hill Yearbook of Science andTechnology (1992) McGraw Hill, New York, N.Y.; pp. 191-196).

An insect system may also be used to express a polypeptide of interest.For example, in one such system, Autographa californica nuclearpolyhedrosis virus (AcNPV) is used as a vector to express foreign genesin Spodoptera frugiperda cells or in Trichoplusia larvae. The sequencesencoding the polypeptide may be cloned into a non-essential region ofthe virus, such as the polyhedrin gene, and placed under control of thepolyhedrin promoter. Successful insertion of the polypeptide-encodingsequence will render the polyhedrin gene inactive and producerecombinant virus lacking coat protein. The recombinant viruses may thenbe used to infect, for example, S. frugiperda cells or Trichoplusialarvae in which the polypeptide of interest may be expressed (Engelhard,E. K. et al. (1994) Proc. Natl. Acad. Sci 91:3224-3227).

In mammalian host cells, a number of viral-based expression systems aregenerally available. For example, in cases where an adenovirus is usedas an expression vector, sequences encoding a polypeptide of interestmay be ligated into an adenovirus transcription/translation complexconsisting of the late promoter and tripartite leader sequence.Insertion in a non-essential E1 or E3 region of the viral genome may beused to obtain a viable virus which is capable of expressing thepolypeptide in infected host cells (Logan, J. and Shenk, T. (1984) Proc.Natl. Acad. Sci. 81:3655-3659). In addition, transcription enhancers,such as the Rous sarcoma virus (RSV) enhancer, may be used to increaseexpression in mammalian host cells.

Specific initiation signals may also be used to achieve more efficienttranslation of sequences encoding a polypeptide of interest. Suchsignals include the ATG initiation codon and adjacent sequences. Incases where sequences encoding the polypeptide, its initiation codon,and upstream sequences are inserted into the appropriate expressionvector, no additional transcriptional or translational control signalsmay be needed. However, in cases where only coding sequence, or aportion thereof, is inserted, exogenous translational control signalsincluding the ATG initiation codon should be provided. Furthermore, theinitiation codon should be in the correct reading frame to ensuretranslation of the entire insert. Exogenous translational elements andinitiation codons may be of various origins, both natural and synthetic.The efficiency of expression may be enhanced by the inclusion ofenhancers which are appropriate for the particular cell system which isused, such as those described in the literature (Scharf, D. et al.(1994) Results Probl. Cell Differ. 20:125-162).

In addition, a host cell strain may be chosen for its ability tomodulate the expression of the inserted sequences or to process theexpressed protein in the desired fashion. Such modifications of thepolypeptide include, but are not limited to, acetylation, carboxylation,glycosylation, phosphorylation, lipidation, and acylation.Post-translational processing which cleaves a “prepro” form of theprotein may also be used to facilitate correct insertion, folding and/orfunction. Different host cells such as CHO, HeLa, MDCK, HEK293, andWI38, which have specific cellular machinery and characteristicmechanisms for such post-translational activities, may be chosen toensure the correct modification and processing of the foreign protein.

For long-term, high-yield production of recombinant proteins, stableexpression is generally preferred. For example, cell lines which stablyexpress a polynucleotide of interest may be transformed using expressionvectors which may contain viral origins of replication and/or endogenousexpression elements and a selectable marker gene on the same or on aseparate vector. Following the introduction of the vector, cells may beallowed to grow for 1-2 days in an enriched media before they areswitched to selective media. The purpose of the selectable marker is toconfer resistance to selection, and its presence allows growth andrecovery of cells which successfully express the introduced sequences.Resistant clones of stably transformed cells may be proliferated usingtissue culture techniques appropriate to the cell type.

Any number of selection systems may be used to recover transformed celllines. These include, but are not limited to, the herpes simplex virusthymidine kinase (Wigler, M. et al. (1977) Cell 11:223-32) and adeninephosphoribosyltransferase (Lowy, I. et al (1990) Cell 22:817-23) geneswhich can be employed in tk.sup.− or aprt.sup.− cells, respectively.Also, antimetabolite, antibiotic or herbicide resistance can be used asthe basis for selection; for example, dhfr which confers resistance tomethotrexate (Wigler, M. et al. (1980) Proc. Natl. Acad. Sci.77:3567-70); npt, which confers resistance to the aminoglycosides,neomycin and G-418 (Colbere-Garapin, F. et al (1981) J. Mol. Biol.150:1-14); and als or pat, which confer resistance to chlorsulfuron andphosphinotricin acetyltransferase, respectively (Murry, supra).Additional selectable genes have been described, for example, trpB,which allows cells to utilize indole in place of tryptophan, or hisD,which allows cells to utilize histinol in place of histidine (Hartman,S. C. and R. C. Mulligan (1988) Proc. Natl. Acad. Sci. 85:8047-51).Recently, the use of visible markers has gained popularity with suchmarkers as anthocyanins, beta-glucuronidase and its substrate GUS, andluciferase and its substrate luciferin, being widely used not only toidentify transformants, but also to quantify the amount of transient orstable protein expression attributable to a specific vector system(Rhodes, C. A. et al. (1995) Methods Mol. Biol. 55:121-131).

Although the presence/absence of marker gene expression suggests thatthe gene of interest is also present, its presence and expression mayneed to be confirmed. For example, if the sequence encoding apolypeptide is inserted within a marker gene sequence, recombinant cellscontaining sequences can be identified by the absence of marker genefunction. Alternatively, a marker gene can be placed in tandem with apolypeptide-encoding sequence under the control of a single promoter.Expression of the marker gene in response to induction or selectionusually indicates expression of the tandem gene as well.

Alternatively, host cells which contain and express a desiredpolynucleotide sequence may be identified by a variety of proceduresknown to those of skill in the art. These procedures include, but arenot limited to, DNA-DNA or DNA-RNA hybridizations and protein bioassayor immunoassay techniques which include membrane, solution, or chipbased technologies for the detection and/or quantification of nucleicacid or protein.

A variety of protocols for detecting and measuring the expression ofpolynucleotide-encoded products, using either polyclonal or monoclonalantibodies specific for the product are known in the art. Examplesinclude enzyme-linked immunosorbent assay (ELISA), radioimmunoassay(RIA), and fluorescence activated cell sorting (FACS). A two-site,monoclonal-based immunoassay utilizing monoclonal antibodies reactive totwo non-interfering epitopes on a given polypeptide may be preferred forsome applications, but a competitive binding assay may also be employed.These and other assays are described, among other places, in Hampton, R.et al. (1990; Serological Methods, a Laboratory Manual, APS Press, StPaul. Minn.) and Maddox, D. E. et al. (1983; J. Exp. Med.158:1211-1216).

A wide variety of labels and conjugation techniques are known by thoseskilled in the art and may be used in various nucleic acid and aminoacid assays. Means for producing labeled hybridization or PCR probes fordetecting sequences related to polynucleotides include oligolabeling,nick translation, end-labeling or PCR amplification using a labelednucleotide. Alternatively, the sequences, or any portions thereof may becloned into a vector for the production of an mRNA probe. Such vectorsare known in the art, are commercially available, and may be used tosynthesize RNA probes in vitro by addition of an appropriate RNApolymerase such as T7, T3, or SP6 and labeled nucleotides. Theseprocedures may be conducted using a variety of commercially availablekits. Suitable reporter molecules or labels, which may be used includeradionuclides, enzymes, fluorescent, chemiluminescent, or chromogenicagents as well as substrates, cofactors, inhibitors, magnetic particles,and the like.

Host cells transformed with a polynucleotide sequence of interest may becultured under conditions suitable for the expression and recovery ofthe protein from cell culture. The protein produced by a recombinantcell may be secreted or contained intracellularly depending on thesequence and/or the vector used. As will be understood by those of skillin the art, expression vectors containing polynucleotides of theinvention may be designed to contain signal sequences which directsecretion of the encoded polypeptide through a prokaryotic or eukaryoticcell membrane. Other recombinant constructions may be used to joinsequences encoding a polypeptide of interest to nucleotide sequenceencoding a polypeptide domain which will facilitate purification ofsoluble proteins. Such purification facilitating domains include, butare not limited to, metal chelating peptides such ashistidine-tryptophan modules that allow purification on immobilizedmetals, protein A domains that allow purification on immobilizedimmunoglobulin, and the domain utilized in the FLAGS extension/affinitypurification system (Immunex Corp., Seattle, Wash.). The inclusion ofcleavable linker sequences such as those specific for Factor XA orenterokinase (Invitrogen. San Diego, Calif.) between the purificationdomain and the encoded polypeptide may be used to facilitatepurification. One such expression vector provides for expression of afusion protein containing a polypeptide of interest and a nucleic acidencoding 6 histidine residues preceding a thioredoxin or an enterokinasecleavage site. The histidine residues facilitate purification on IMIAC(immobilized metal ion affinity chromatography) as described in Porath,J. et al. (1992, Prot. Exp. Purif. 3:263-281) while the enterokinasecleavage site provides a means for purifying the desired polypeptidefrom the fusion protein. A discussion of vectors which contain fusionproteins is provided in Kroll, D. J. et al. (1993; DNA Cell Biol.12:441-453).

In addition to recombinant production methods, polypeptides of theinvention, and fragments thereof, may be produced by direct peptidesynthesis using solid-phase techniques (Merrifield J. (1963) J. Am.Chem. Soc. 85:2149-2154). Protein synthesis may be performed usingmanual techniques or by automation. Automated synthesis may be achieved,for example, using Applied Biosystems 431A Peptide Synthesizer (PerkinElmer). Alternatively, various fragments may be chemically synthesizedseparately and combined using chemical methods to produce the fulllength molecule.

4.13 Site-Specific Mutagenesis

Site-specific mutagenesis is a technique useful in the preparation ofindividual peptides, or biologically functional equivalent polypeptides,through specific mutagenesis of the underlying polynucleotides thatencode them. The technique, well-known to those of skill in the art,further provides a ready ability to prepare and test sequence variants,for example, incorporating one or more of the foregoing considerations,by introducing one or more nucleotide sequence changes into the DNA.Site-specific mutagenesis allows the production of mutants through theuse of specific oligonucleotide sequences which encode the DNA sequenceof the desired mutation, as well as a sufficient number of adjacentnucleotides, to provide a primer sequence of sufficient size andsequence complexity to form a stable duplex on both sides of thedeletion junction being traversed. Mutations may be employed in aselected polynucleotide sequence to improve, alter, decrease, modify, orotherwise change the properties of the polynucleotide itself, and/oralter the properties, activity, composition, stability, or primarysequence of the encoded polypeptide.

In certain embodiments of the present invention, the inventorscontemplate the mutagenesis of the disclosed polynucleotide sequences toalter one or more properties of the encoded polypeptide, such as theantigenicity of a polypeptide vaccine. The techniques of site-specificmutagenesis are well-known in the art, and are widely used to createvariants of both polypeptides and polynucleotides. For example,site-specific mutagenesis is often used to alter a specific portion of aDNA molecule. In such embodiments, a primer comprising typically about14 to about 25 nucleotides or so in length is employed, with about 5 toabout 10 residues on both sides of the junction of the sequence beingaltered.

As will be appreciated by those of skill in the art, site-specificmutagenesis techniques have often employed a phage vector that exists inboth a single stranded and double stranded form. Typical vectors usefulin site-directed mutagenesis include vectors such as the M13 phage.These phage are readily commercially-available and their use isgenerally well-known to those skilled in the art. Double-strandedplasmids are also routinely employed in site directed mutagenesis thateliminates the step of transferring the gene of interest from a plasmidto a phage.

In general, site-directed mutagenesis in accordance herewith isperformed by first obtaining a single-stranded vector or melting apartof two strands of a double-stranded vector that includes within itssequence a DNA sequence that encodes the desired peptide. Anoligonucleotide primer bearing the desired mutated sequence is prepared,generally synthetically. This primer is then annealed with thesingle-stranded vector, and subjected to DNA polymerizing enzymes suchas E. coli polymerase I Klenow fragment, in order to complete thesynthesis of the mutation-bearing strand. Thus, a heteroduplex is formedwherein one strand encodes the original non-mutated sequence and thesecond strand bears the desired mutation. This heteroduplex vector isthen used to transform appropriate cells, such as E. coli cells, andclones are selected which include recombinant vectors bearing themutated sequence arrangement.

The preparation of sequence variants of the selected peptide-encodingDNA segments using site-directed mutagenesis provides a means ofproducing potentially useful species and is not meant to be limiting asthere are other ways in which sequence variants of peptides and the DNAsequences encoding them may be obtained. For example, recombinantvectors encoding the desired peptide sequence may be treated withmutagenic agents, such as hydroxylamine, to obtain sequence variants.Specific details regarding these methods and protocols are found in theteachings of Maloy et al., 1994; Segal, 1976; Prokop and Bajpai, 1991;Kuby, 1994; and Maniatis et al., 1982, each incorporated herein byreference, for that purpose.

As used herein, the term “oligonucleotide directed mutagenesisprocedure” refers to template-dependent processes and vector-mediatedpropagation which result in an increase in the concentration of aspecific nucleic acid molecule relative to its initial concentration, orin an increase in the concentration of a detectable signal, such asamplification. As used herein, the term “oligonucleotide directedmutagenesis procedure” is intended to refer to a process that involvesthe template-dependent extension of a primer molecule. The term templatedependent process refers to nucleic acid synthesis of an RNA or a DNAmolecule wherein the sequence of the newly synthesized strand of nucleicacid is dictated by the well-known rules of complementary base pairing(see, for example, Watson, 1987). Typically, vector mediatedmethodologies involve the introduction of the nucleic acid fragment intoa DNA or RNA vector, the clonal amplification of the vector, and therecovery of the amplified nucleic acid fragment. Examples of suchmethodologies are provided by U.S. Pat. No. 4,237,224, specificallyincorporated herein by reference in its entirety.

4.14 Polynucleotide Amplification Techniques

A number of template dependent processes are available to amplify thetarget sequences of interest present in a sample. One of the best knownamplification methods is the polymerase chain reaction (PCR™) which isdescribed in detail in U.S. Pat. Nos. 4,683,195, 4,683,202 and4,800,159, each of which is incorporated herein by reference in itsentirety. Briefly, in PCR™, two primer sequences are prepared which arecomplementary to regions on opposite complementary strands of the targetsequence. An excess of deoxynucleoside triphosphates is added to areaction mixture along with a DNA polymerase (e.g., Taq polymerase). Ifthe target sequence is present in a sample, the primers will bind to thetarget and the polymerase will cause the primers to be extended alongthe target sequence by adding on nucleotides. By raising and loweringthe temperature of the reaction mixture, the extended primers willdissociate from the target to form reaction products, excess primerswill bind to the target and to the reaction product and the process isrepeated. Preferably reverse transcription and PCR™ amplificationprocedure may be performed in order to quantify the amount of mRNAamplified. Polymerase chain reaction methodologies are well known in theart.

Another method for amplification is the ligase chain reaction (referredto as LCR), disclosed in Eur. Pat. Appl. Publ. No. 320,308 (specificallyincorporated herein by reference in its entirety). In LCR, twocomplementary probe pairs are prepared, and in the presence of thetarget sequence, each pair will bind to opposite complementary strandsof the target such that they abut. In the presence of a ligase, the twoprobe pairs will link to form a single unit. By temperature cycling, asin PCR™, bound ligated units dissociate from the target and then serveas “target sequences” for ligation of excess probe pairs. U.S. Pat. No.4,883,750, incorporated herein by reference in its entirety, describesan alternative method of amplification similar to LCR for binding probepairs to a target sequence.

Qbeta Replicase, described in PCT Intl. Pat. Appl. Publ. No.PCT/US87/00880, incorporated herein by reference in its entirety, mayalso be used as still another amplification method in the presentinvention. In this method, a replicative sequence of RNA that has aregion complementary to that of a target is added to a sample in thepresence of an RNA polymerase. The polymerase will copy the replicativesequence that can then be detected.

An isothermal amplification method, in which restriction endonucleasesand ligases are used to achieve the amplification of target moleculesthat contain nucleotide 5′-[α-thio]triphosphates in one strand of arestriction site (Walker et al., 1992, incorporated herein by referencein its entirety), may also be useful in the amplification of nucleicacids in the present invention.

Strand Displacement Amplification (SDA) is another method of carryingout isothermal amplification of nucleic acids which involves multiplerounds of strand displacement and synthesis, i.e. nick translation. Asimilar method, called Repair Chain Reaction (RCR) is another method ofamplification which may be useful in the present invention and isinvolves annealing several probes throughout a region targeted foramplification, followed by a repair reaction in which only two of thefour bases are present. The other two bases can be added as biotinylatedderivatives for easy detection. A similar approach is used in SDA.

Sequences can also be detected using a cyclic probe reaction (CPR). InCPR, a probe having a 3′ and 5′ sequences of non-target DNA and aninternal or “middle” sequence of the target protein specific RNA ishybridized to DNA which is present in a sample. Upon hybridization, thereaction is treated with RNaseH, and the products of the probe areidentified as distinctive products by generating a signal that isreleased after digestion. The original template is annealed to anothercycling probe and the reaction is repeated. Thus, CPR involvesamplifying a signal generated by hybridization of a probe to a targetgene specific expressed nucleic acid.

Still other amplification methods described in Great Britain Pat. Appl.No. 2 202 328, and in PCT Intl. Pat. Appl. Publ. No. PCT/US89/01025,each of which is incorporated herein by reference in its entirety, maybe used in accordance with the present invention. In the formerapplication, “modified” primers are used in a PCR-like, template andenzyme dependent synthesis. The primers may be modified by labeling witha capture moiety (e.g. biotin) and/or a detector moiety (e.g., enzyme).In the latter application, an excess of labeled probes is added to asample. In the presence of the target sequence, the probe binds and iscleaved catalytically. After cleavage, the target sequence is releasedintact to be bound by excess probe. Cleavage of the labeled probesignals the presence of the target sequence.

Other nucleic acid amplification procedures include transcription-basedamplification systems (TAS) (Kwoh et al., 1989; PCT Intl. Pat. Appl.Publ. No. WO 88/10315, incorporated herein by reference in itsentirety), including nucleic acid sequence based amplification (NASBA)and 3SR. In NASBA, the nucleic acids can be prepared for amplificationby standard phenol/chloroform extraction, heat denaturation of a sample,treatment with lysis buffer and minispin columns for isolation of DNAand RNA or guanidinium chloride extraction of RNA. These amplificationtechniques involve annealing a primer that has sequences specific to thetarget sequence. Following polymerization, DNA/RNA hybrids are digestedwith RNase H while double stranded DNA molecules are heat-denaturedagain. In either case the single stranded DNA is made fully doublestranded by addition of second target-specific primer, followed bypolymerization. The double stranded DNA molecules are then multiplytranscribed by a polymerase such as T7 or SP6. In an isothermal cyclicreaction, the RNAs are reverse transcribed into DNA, and transcribedonce again with a polymerase such as T7 or SP6. The resulting products,whether truncated or complete, indicate target-specific sequences.

Eur. Pat. Appl. Publ. No. 329,822, incorporated herein by reference inits entirety, disclose a nucleic acid amplification process involvingcyclically synthesizing single-stranded RNA (“ssRNA”), ssDNA, anddouble-stranded DNA (dsDNA), which may be used in accordance with thepresent invention. The ssRNA is a first template for a first primeroligonucleotide, which is elongated by reverse transcriptase(RNA-dependent DNA polymerase). The RNA is then removed from resultingDNA:RNA duplex by the action of ribonuclease H(RNase H, an RNasespecific for RNA in a duplex with either DNA or RNA). The resultantssDNA is a second template for a second primer, which also includes thesequences of an RNA polymerase promoter (exemplified by T7 RNApolymerase) 5′ to its homology to its template. This primer is thenextended by DNA polymerase (exemplified by the large “Klenow” fragmentof E. coli DNA polymerase I), resulting as a double-stranded DNA(“dsDNA”) molecule, having a sequence identical to that of the originalRNA between the primers and having additionally, at one end, a promotersequence. This promoter sequence can be used by the appropriate RNApolymerase to make many RNA copies of the DNA. These copies can thenre-enter the cycle leading to very swift amplification. With properchoice of enzymes, this amplification can be done isothermally withoutaddition of enzymes at each cycle. Because of the cyclical name of thisprocess, the starting sequence can be chosen to be in the form of eitherDNA or RNA.

PCT Intl. Pat. Appl. Publ. No. WO 89/06700, incorporated herein byreference in its entirety, disclose a nucleic acid sequenceamplification scheme based on the hybridization of a promoter/primersequence to a target single-stranded DNA (“ssDNA”) followed bytranscription of many RNA copies of the sequence. This scheme is notcyclic; i.e. new templates are not produced from the resultant RNAtranscripts. Other amplification methods include “RACE” (Frohman, 1990),and “one-sided PCR” (Ohara, 1989) which are well-known to those of skillin the art.

Methods based on ligation of two (or more) oligonucleotides in thepresence of nucleic acid having the sequence of the resulting“di-oligonucleotide”, thereby amplifying the di-oligonucleotide (Wu andDean, 1996, incorporated herein by reference in its entirety), may alsobe used in the amplification of DNA sequences of the present invention.

4.15 In Vivo Polynucleotide Delivery Techniques

In additional embodiments, genetic constructs comprising one or more ofthe polynucleotides of the invention are introduced into cells in vivo.This may be achieved using any of a variety or well known approaches,several of which are outlined below for the purpose of illustration.

4.15.1 Adenovirus

One of the preferred methods for in vivo delivery of one or more nucleicacid sequences involves the use of an adenovirus expression vector.“Adenovirus expression vector” is meant to include those constructscontaining adenovirus sequences sufficient to (a) support packaging ofthe construct and (b) to express a polynucleotide that has been clonedtherein in a sense or antisense orientation. Of course, in the contextof an antisense construct, expression does not require that the geneproduct be synthesized.

The expression vector comprises a genetically engineered form of anadenovirus. Knowledge of the genetic organization of adenovirus, a 36kb, linear, double-stranded DNA virus, allows substitution of largepieces of adenoviral DNA with foreign sequences up to 7 kb (Grunhaus andHorwitz, 1992). In contrast to retrovirus, the adenoviral infection ofhost cells does not result in chromosomal integration because adenoviralDNA can replicate in an episomal manner without potential genotoxicity.Also, adenoviruses are structurally stable, and no genome rearrangementhas been detected after extensive amplification. Adenovirus can infectvirtually all epithelial cells regardless of their cell cycle stage. Sofar, adenoviral infection appears to be linked only to mild disease suchas acute respiratory disease in humans.

Adenovirus is particularly suitable for use as a gene transfer vectorbecause of its mid-sized genome, ease of manipulation, high titer, widetarget-cell range and high infectivity. Both ends of the viral genomecontain 100-200 base pair inverted repeats (ITRs), which are ciselements necessary for viral DNA replication and packaging. The early(E) and late (L) regions of the genome contain different transcriptionunits that are divided by the onset of viral DNA replication. The E1region (E1A and E1B) encodes proteins responsible for the regulation oftranscription of the viral genome and a few cellular genes. Theexpression of the E2 region (E2A and E2B) results in the synthesis ofthe proteins for viral DNA replication. These proteins are involved inDNA replication, late gene expression and host cell shut-off (Renan,1990). The products of the late genes, including the majority of theviral capsid proteins, are expressed only after significant processingof a single primary transcript issued by the major late promoter (MLP).The MLP, (located at 16.8 m.u.) is particularly efficient during thelate phase of infection, and all the mRNA's issued from this promoterpossess a 5′-tripartite leader (TPL) sequence which makes them preferredmRNA's for translation.

In a current system, recombinant adenovirus is generated from homologousrecombination between shuttle vector and provirus vector. Due to thepossible recombination between two proviral vectors, wild-typeadenovirus may be generated from this process. Therefore, it is criticalto isolate a single clone of virus from an individual plaque and examineits genomic structure.

Generation and propagation of the current adenovirus vectors, which arereplication deficient, depend on a unique helper cell line, designated293, which was transformed from human embryonic kidney cells by Ad5 DNAfragments and constitutively expresses E1 proteins (Graham et al.,1977). Since the E3 region is dispensable from the adenovirus genome(Jones and Shenk, 1978), the current adenovirus vectors, with the helpof 293 cells, carry foreign DNA in either the E1, the D3 or both regions(Graham and Prevec, 1991). In nature, adenovirus can packageapproximately 105% of the wild-type genome (Ghosh-Choudhury et al.,1987), providing capacity for about 2 extra kB of DNA Combined with theapproximately 5.5 kB of DNA that is replaceable in the E1 and E3regions, the maximum capacity of the current adenovirus vector is under7.5 kB, or about 15% of the total length of the vector. More than 80% ofthe adenovirus viral genome remains in the vector backbone and is thesource of vector-borne cytotoxicity. Also, the replication deficiency ofthe E1-deleted virus is incomplete. For example, leakage of viral geneexpression has been observed with the currently available vectors athigh multiplicities of infection (MOI) (Mulligan, 1993).

Helper cell lines may be derived from human cells such as humanembryonic kidney cells, muscle cells, hematopoietic cells or other humanembryonic mesenchymal or epithelial cells. Alternatively, the helpercells may be derived from the cells of other mammalian species that arepermissive for human adenovirus. Such cells include, e.g., Vero cells orother monkey embryonic mesenchymal or epithelial cells. As stated above,the currently preferred helper cell line is 293.

Recently, Racher et al. (1995) disclosed improved methods for culturing293 cells and propagating adenovirus. In one format, natural cellaggregates are grown by inoculating individual cells into 1 litersiliconized spinner flasks (Techne, Cambridge, UK) containing 100-200 mlof medium. Following stirring at 40 rpm, the cell viability is estimatedwith trypan blue. In another format, Fibra-Cel microcarriers (BibbySterlin, Stone, UK) (5 g/l) is employed as follows. A cell inoculum,resuspended in 5 ml of medium, is added to the carrier (50 ml) in a 250ml Erlenmeyer flask and left stationary, with occasional agitation, for1 to 4 h. The medium is then replaced with 50 ml of fresh medium andshaking initiated. For virus production, cells are allowed to grow toabout 80% confluence, after which time the medium is replaced (to 25% ofthe final volume) and adenovirus added at an MOI of 0.05. Cultures areleft stationary overnight, following which the volume is increased to100% and shaking commenced for another 72 h.

Other than the requirement that the adenovirus vector be replicationdefective, or at least conditionally defective, the nature of theadenovirus vector is not believed to be crucial to the successfulpractice of the invention. The adenovirus may be of any of the 42different known serotypes or subgroups A-F. Adenovirus type 5 ofsubgroup C is the preferred starting material in order to obtain aconditional replication-defective adenovirus vector for use in thepresent invention, since Adenovirus type 5 is a human adenovirus aboutwhich a great deal of biochemical and genetic information is known, andit has historically been used for most constructions employingadenovirus as a vector.

As stated above, the typical vector according to the present inventionis replication defective and will not have an adenovirus E1 region.Thus, it will be most convenient to introduce the polynucleotideencoding the gene of interest at the position from which the E1-codingsequences have been removed. However, the position of insertion of theconstruct within the adenovirus sequences is not critical to theinvention. The polynucleotide encoding the gene of interest may also beinserted in lieu of the deleted E3 region in E3 replacement vectors asdescribed by Karlsson et al. (1986) or in the E4 region where a helpercell line or helper virus complements the E4 defect.

Adenovirus is easy to grow and manipulate and exhibits broad host rangein vitro and in vivo. This group of viruses can be obtained in hightiters, e.g., 10⁹-10¹¹ plaque-forming units per ml, and they are highlyinfective. The life cycle of adenovirus does not require integrationinto the host cell genome. The foreign genes delivered by adenovirusvectors are episomal and, therefore, have low genotoxicity to hostcells. No side effects have been reported in studies of vaccination withwild-type adenovirus (Couch et al., 1963; Top et al., 1971),demonstrating their safety and therapeutic potential as in vivo genetransfer vectors.

Adenovirus vectors have been used in eukaryotic gene expression (Levreroet al., 1991; Gomez-Foix et al., 1992) and vaccine development (Grunhausand Horwitz, 1992; Graham and Prevec, 1992). Recently, animal studiessuggested that recombinant adenovirus could be used for gene therapy(Stratford-Perricaudet and Perricaudet, 1991; Stratford-Perricaudet etal., 1990; Rich et al., 1993). Studies in administering recombinantadenovirus to different tissues include trachea instillation (Rosenfeldet al., 1991; Rosenfeld et al., 1992), muscle injection (Ragot et al.,1993), peripheral intravenous injections (Herz and Gerard, 1993) andstereotactic inoculation into the brain (Le Gal La Salle et al., 1993).

4.15.2 Retroviruses

The retroviruses are a group of single-stranded RNA virusescharacterized by an ability to convert their RNA to double-stranded DNAin infected cells by a process of reverse-transcription (Coffin, 1990).The resulting DNA then stably integrates into cellular chromosomes as aprovirus and directs synthesis of viral proteins. The integrationresults in the retention of the viral gene sequences in the recipientcell and its descendants. The retroviral genome contains three genes,gag, pol, and env that code for capsid proteins, polymerase enzyme, andenvelope components, respectively. A sequence found upstream from thegag gene contains a signal for packaging of the genome into virions. Twolong terminal repeat (LTR) sequences are present at the 5′ and 3′ endsof the viral genome. These contain strong promoter and enhancersequences and are also required for integration in the host cell genome(Coffin, 1990).

In order to construct a retroviral vector, a nucleic acid encoding oneor more oligonucleotide or polynucleotide sequences of interest isinserted into the viral genome in the place of certain viral sequencesto produce a virus that is replication-defective. In order to producevirions, a packaging cell line containing the gag, pol, and env genesbut without the LTR and packaging components is constructed (Mann etal., 1983). When a recombinant plasmid containing a cDNA, together withthe retroviral LTR and packaging sequences is introduced into this cellline (by calcium phosphate precipitation for example), the packagingsequence allows the RNA transcript of the recombinant plasmid to bepackaged into viral particles, which are then secreted into the culturemedia (Nicolas and Rubenstein, 1988; Temin, 1986; Mann et al., 1983).The media containing the recombinant retroviruses is then collected,optionally concentrated, and used for gene transfer. Retroviral vectorsare able to infect a broad variety of cell types. However, integrationand stable expression require the division of host cells (Paskind etal., 1975).

A novel approach designed to allow specific targeting of retrovirusvectors was recently developed based on the chemical modification of aretrovirus by the chemical addition of lactose residues to the viralenvelope. This modification could permit the specific infection ofhepatocytes via sialoglycoprotein receptors.

A different approach to targeting of recombinant retroviruses wasdesigned in which biotinylated antibodies against a retroviral envelopeprotein and against a specific cell receptor were used. The antibodieswere coupled via the biotin components by using streptavidin (Roux etal., 1989). Using antibodies against major histocompatibility complexclass I and class II antigens, they demonstrated the infection of avariety of human cells that bore those surface antigens with anecotropic virus in vitro Roux et al., 1989).

4.15.3 Adeno-Associated Viruses

AAV (Ridgeway, 1988; Hermonat and Muzycska, 1984) is a parovirus,discovered as a contamination of adenoviral stocks. It is a ubiquitousvirus (antibodies are present in 85% of the US human population) thathas not been linked to any disease. It is also classified as adependovirus, because its replications is dependent on the presence of ahelper virus, such as adenovirus. Five serotypes have been isolated, ofwhich AAV-2 is the best characterized. AAV has a single-stranded linearDNA that is encapsidated into capsid proteins VP1, VP2 and VP3 to forman icosahedral virion of 20 to 24 nm in diameter (Muzyczka andMcLaughlin, 1988).

The AAV DNA is approximately 4.7 kilobases long. It contains two openreading frames and is flanked by two ITRs. There are two major genes inthe AAV genome: rep and cap. The rep gene codes for proteins responsiblefor viral replications, whereas cap codes for capsid protein VP1-3. EachITR forms a T-shaped hairpin structure. These terminal repeats are theonly essential cis components of the AAV for chromosomal integration.Therefore, the AAV can be used as a vector with all viral codingsequences removed and replaced by the cassette of genes for delivery.Three viral promoters have been identified and named p5, p19, and p40,according to their map position. Transcription from p5 and p19 resultsin production of rep proteins, and transcription from p40 produces thecapsid proteins (Hermonat and Muzyczka, 1984).

There are several factors that prompted researchers to study thepossibility of using rAAV as an expression vector. One is that therequirements for delivering a gene to integrate into the host chromosomeare surprisingly few. It is necessary to have the 145-bp ITRs, which areonly 6% of the AAV genome. This leaves room in the vector to assemble a4.5-kb DNA insertion. While this carrying capacity may prevent the AAVfrom delivering large genes, it is amply suited for delivering theantisense constructs of the present invention AAV is also a good choiceof delivery vehicles due to its safety. There is a relativelycomplicated rescue mechanism: not only wild type adenovirus but also AAVgenes are required to mobilize rAAV. Likewise, AAV is not pathogenic andnot associated with any disease. The removal of viral coding sequencesminimizes immune reactions to viral gene expression, and therefore, rAAVdoes not evoke an inflammatory response.

4.15.4 Other Viral Vectors as Expression Constructs

Other viral vectors may be employed as expression constructs in thepresent invention for the delivery of oligonucleotide or polynucleotidesequences to a host cell. Vectors derived from viruses such as vacciniavirus (Ridgeway, 1988; Coupar et al., 1988), lentiviruses, polio virusesand herpes viruses may be employed. They offer several attractivefeatures for various mammalian cells (Friedmann, 1989; Ridgeway, 1988;Coupar et al., 1988; Horwich et al., 1990).

With the recent recognition of defective hepatitis B viruses, newinsight was gained into the structure-function relationship of differentviral sequences. In vitro studies showed that the virus could retain theability for helper-dependent packaging and reverse transcription despitethe deletion of up to 80% of its genome (Horwich et al., 1990). Thissuggested that large portions of the genome could be replaced withforeign genetic material. The hepatotropism and persistence(integration) were particularly attractive properties for liver-directedgene transfer. Chang et al. (1991) introduced the chloramphenicolacetyltransferase (CAT) gene into duck hepatitis B virus genome in theplace of the polymerase, surface, and pre-surface coding sequences. Itwas cotransfected with wild-type virus into an avian hepatoma cell line.Culture media containing high titers of the recombinant virus were usedto infect primary duckling hepatocytes. Stable CAT gene expression wasdetected for at least 24 days after transfection (Chang et al., 1991).

4.15.5 Non-Viral Vectors

In order to effect expression of the oligonucleotide or polynucleotidesequences of the present invention, the expression construct must bedelivered into a cell. This delivery may be accomplished in vitro, as inlaboratory procedures for transforming cells lines, or in vivo or exvivo, as in the treatment of certain disease states. As described above,one preferred mechanism for delivery is via viral infection where theexpression construct is encapsulated in an infectious viral particle.

Once the expression construct has been delivered into the cell thenucleic acid encoding the desired oligonucleotide or polynucleotidesequences may be positioned and expressed at different sites. In certainembodiments, the nucleic acid encoding the construct may be stablyintegrated into the genome of the cell. This integration may be in thespecific location and orientation via homologous recombination (genereplacement) or it may be integrated in a random, non-specific location(gene augmentation). In yet further embodiments, the nucleic acid may bestably maintained in the cell as a separate, episomal segment of DNA.Such nucleic acid segments or “episomes” encode sequences sufficient topermit maintenance and replication independent of or in synchronizationwith the host cell cycle. How the expression construct is delivered to acell and where in the cell the nucleic acid remains is dependent on thetype of expression construct employed.

In certain embodiments of the invention, the expression constructcomprising one or more oligonucleotide or polynucleotide sequences maysimply consist of naked recombinant DNA or plasmids. Transfer of theconstruct may be performed by any of the methods mentioned above whichphysically or chemically permeabilize the cell membrane. This isparticularly applicable for transfer in vitro but it may be applied toin vivo use as well. Dubensky et al. (1984) successfully injectedpolyomavirus DNA in the form of calcium phosphate precipitates intoliver and spleen of adult and newborn mice demonstrating active viralreplication and acute infection. Benvenisty and Reshef (1986) alsodemonstrated that direct intraperitoneal injection of calciumphosphate-precipitated plasmids results in expression of the transfectedgenes. It is envisioned that DNA encoding a gene of interest may also betransferred in a similar manner in vivo and express the gene product.

Another embodiment of the invention for transferring a naked DNAexpression construct into cells may involve particle bombardment. Thismethod depends on the ability to accelerate DNA-coated microprojectilesto a high velocity allowing them to pierce cell membranes and entercells without killing them (Klein et al., 1987). Several devices foraccelerating small particles have been developed. One such device relieson a high voltage discharge to generate an electrical current, which inturn provides the motive force (Yang et al., 1990). The microprojectilesused have consisted of biologically inert substances such as tungsten orgold beads.

Selected organs including the liver, skin, and muscle tissue of rats andmice have been bombarded in vivo (Yang et al., 1990; Zelenin et al.,1991). This may require surgical exposure of the tissue or cells, toeliminate any intervening tissue between the gun and the target organ,i.e. ex vivo treatment. Again, DNA encoding a particular gene may bedelivered via this method and still be incorporated by the presentinvention.

4.16 Antisense Oligonucleotides

The end result of the flow of genetic information is the synthesis ofprotein. DNA is transcribed by polymerases into messenger RNA andtranslated on the ribosome to yield a folded, functional protein. Thusthere are several steps along the route where protein synthesis can beinhibited. The native DNA segment coding for a polypeptide describedherein, as all such mammalian DNA strands, has two strands: a sensestrand and an antisense strand held together by hydrogen bonding. Themessenger RNA coding for polypeptide has the same nucleotide sequence asthe sense DNA strand except that the DNA thymidine is replaced byuridine. Thus, synthetic antisense nucleotide sequences will bind to amRNA and inhibit expression of the protein encoded by that mRNA.

The targeting of antisense oligonucleotides to mRNA is thus onemechanism to shut down protein synthesis, and, consequently, representsa powerful and targeted therapeutic approach. For example, the synthesisof polygalactauronase and the muscarine type 2 acetylcholine receptorare inhibited by antisense oligonucleotides directed to their respectivemRNA sequences (U.S. Pat. No. 5,739,119 and U.S. Pat. No. 5,759,829,each specifically incorporated herein by reference in its entirety).Further, examples of antisense inhibition have been demonstrated withthe nuclear protein cyclin, the multiple drug resistance gene (MDG1),ICAM-1, E-selectin, STK-1, striatal GABA_(A) receptor and human EGF(Jaskulski et al., 1988; Vasanthakumar and Ahmed, 1989; Peris et al.,1998; U.S. Pat. No. 5,801,154; U.S. Pat. No. 5,789,573; U.S. Pat. No.5,718,709 and U.S. Pat. No. 5,610,288, each specifically incorporatedherein by reference in its entirety). Antisense constructs have alsobeen described that inhibit and can be used to treat a variety ofabnormal cellular proliferations, e.g. cancer (U.S. Pat. No. 5,747,470;U.S. Pat. No. 5,591,317 and U.S. Pat. No. 5,783,683, each specificallyincorporated herein by reference in its entirety).

Therefore, in exemplary embodiments, the invention providesoligonucleotide sequences that comprise all, or a portion of, anysequence that is capable of specifically binding to polynucleotidesequence described herein, or a complement thereof. In one embodiment,the antisense oligonucleotides comprise DNA or derivatives thereof. Inanother embodiment, the oligonucleotides comprise RNA or derivativesthereof. In a third embodiment, the oligonucleotides are modified DNAscomprising a phosphorothioated modified backbone. In a fourthembodiment, the oligonucleotide sequences comprise peptide nucleic acidsor derivatives thereof. In each case, preferred compositions comprise asequence region that is complementary, and more preferablysubstantially-complementary, and even more preferably, completelycomplementary to one or more portions of polynucleotides disclosedherein.

Selection of antisense compositions specific for a given gene sequenceis based upon analysis of the chosen target sequence (i.e. in theseillustrative examples the rat and human sequences) and determination ofsecondary structure, T_(m), binding energy, relative stability, andantisense compositions were selected based upon their relative inabilityto form dimers, hairpins, or other secondary structures that wouldreduce or prohibit specific binding to the target mRNA in a host cell.

Highly preferred target regions of the mRNA, are those which are at ornear the AUG translation initiation codon, and those sequences whichwere substantially complementary to 5′ regions of the mRNA. Thesesecondary structure analyses and target site selection considerationswere performed using v.4 of the OLIGO primer analysis software (Rychlik,1997) and the BLASTN 2.0.5 algorithm software (Altschul et al., 1997).

The use of an antisense delivery method employing a short peptidevector, termed MPG (27 residues), is also contemplated. The MPG peptidecontains a hydrophobic domain derived from the fusion sequence of HIVgp41 and a hydrophilic domain from the nuclear localization sequence ofSV40 T-antigen (Morris et al., 1997). It has been demonstrated thatseveral molecules of the MPG peptide coat the antisense oligonucleotidesand can be delivered into cultured mammalian cells in less than 1 hourwith relatively high efficiency (90%). Further, the interaction with MPGstrongly increases both the stability of the oligonucleotide to nucleaseand the ability to cross the plasma membrane (Morris et al., 1997).

4.17 Ribozymes

Although proteins traditionally have been used for catalysis of nucleicacids, another class of macromolecules has emerged as useful in thisendeavor. Ribozymes are RNA-protein complexes that cleave nucleic acidsin a site-specific fashion. Ribozymes have specific catalytic domainsthat possess endonuclease activity (Kim and Cech, 1987; Gerlach et al.,1987; Forster and Symons, 1987). For example, a large number ofribozymes accelerate phosphoester transfer reactions with a high degreeof specificity, often cleaving only one of several phosphoesters in anoligonucleotide substrate (Cech et al., 1981; Michel and Westhof, 1990;Reinhold-Hurek and Shub, 1992). This specificity has been attributed tothe requirement that the substrate bind via specific base-pairinginteractions to the internal guide sequence (“IGS”) of the ribozymeprior to chemical reaction.

Ribozyme catalysis has primarily been observed as part ofsequence-specific cleavage/ligation reactions involving nucleic acids(Joyce, 1989; Cech et al., 1981). For example, U.S. Pat. No. 5,354,855(specifically incorporated herein by reference) reports that certainribozymes can act as endonucleases with a sequence specificity greaterthan that of known ribonucleases and approaching that of the DNArestriction enzymes. Thus, sequence-specific ribozyme-mediatedinhibition of gene expression may be particularly suited to therapeuticapplications (Scanlon et al., 1991; Sarver et al., 1990). Recently, itwas reported that ribozymes elicited genetic changes in some cells linesto which they were applied; the altered genes included the oncogenesH-ras, c-fos and genes of HIV. Most of this work involved themodification of a target mRNA, based on a specific mutant codon that iscleaved by a specific ribozyme.

Six basic varieties of naturally-occurring enzymatic RNAs are knownpresently. Each can catalyze the hydrolysis of RNA phosphodiester bondsin trans (and thus can cleave other RNA molecules) under physiologicalconditions. In general, enzymatic nucleic acids act by first binding toa target RNA. Such binding occurs through the target binding portion ofa enzymatic nucleic acid which is held in close proximity to anenzymatic portion of the molecule that acts to cleave the target RNA.Thus, the enzymatic nucleic acid first recognizes and then binds atarget RNA through complementary base-pairing, and once bound to thecorrect site, acts enzymatically to cut the target RNA. Strategiccleavage of such a target RNA will destroy its ability to directsynthesis of an encoded protein. After an enzymatic nucleic acid hasbound and cleaved its RNA target, it is released from that RNA to searchfor another target and can repeatedly bind and cleave new targets.

The enzymatic nature of a ribozyme is advantageous over manytechnologies, such as antisense technology (where a nucleic acidmolecule simply binds to a nucleic acid target to block its translation)since the concentration of ribozyme necessary to affect a therapeutictreatment is lower than that of an antisense oligonucleotide. Thisadvantage reflects the ability of the ribozyme to act enzymatically.Thus, a single ribozyme molecule is able to cleave many molecules oftarget RNA. In addition, the ribozyme is a highly specific inhibitor,with the specificity of inhibition depending not only on the basepairing mechanism of binding to the target RNA, but also on themechanism of target RNA cleavage. Single mismatches, orbase-substitutions, near the site of cleavage can completely eliminatecatalytic activity of a ribozyme. Similar mismatches in antisensemolecules do not prevent their action (Woolf et al., 1992). Thus, thespecificity of action of a ribozyme is greater than that of an antisenseoligonucleotide binding the same RNA site.

The enzymatic nucleic acid molecule may be formed in a hammerhead,hairpin, a hepatitis δ virus, group I intron or RNaseP RNA (inassociation with an RNA guide sequence) or Neurospora VS RNA motif.Examples of hammerhead motifs are described by Rossi et al (1992).Examples of hairpin motifs are described by Hampel et al. (Eur. Pat.Appl. Publ. No. EP 0360257), Hampel and Tritz (1989), Hampel et al.(1990) and U.S. Pat. No. 5,631,359 (specifically incorporated herein byreference). An example of the hepatitis δ virus motif is described byPerrotta and Been (1992); an example of the RNaseP motif is described byGuerrier-Takada et al. (1983); Neurospora VS RNA ribozyme motif isdescribed by Collins (Saville and Collins, 1990; Saville and Collins,1991; Collins and Olive, 1993); and an example of the Group I intron isdescribed in (U.S. Pat. No. 4,987,071, specifically incorporated hereinby reference). All that is important in an enzymatic nucleic acidmolecule of this invention is that it has a specific substrate bindingsite which is complementary to one or more of the target gene RNAregions, and that it have nucleotide sequences within or surroundingthat substrate binding site which impart an RNA cleaving activity to themolecule. Thus the ribozyme constructs need not be limited to specificmotifs mentioned herein.

In certain embodiments, it may be important to produce enzymaticcleaving agents which exhibit a high degree of specificity for the RNAof a desired target, such as one of the sequences disclosed herein. Theenzymatic nucleic acid molecule is preferably targeted to a highlyconserved sequence region of a target mRNA. Such enzymatic nucleic acidmolecules can be delivered exogenously to specific cells as required.Alternatively, the ribozymes can be expressed from DNA or RNA vectorsthat are delivered to specific cells.

Small enzymatic nucleic acid motifs (e.g., of the hammerhead or thehairpin structure) may also be used for exogenous delivery. The simplestructure of these molecules increases the ability of the enzymaticnucleic acid to invade targeted regions of the mRNA structure.Alternatively, catalytic RNA molecules can be expressed within cellsfrom eukaryotic promoters (e.g., Scanlon et al, 1991; Kashani-Sabet etal, 1992; Dropulic et al., 1992; Weerasinghe et al., 1991; Ojwang etal., 1992; Chen et al., 1992; Sarver et al., 1990). Those skilled in theart realize that any ribozyme can be expressed in eukaryotic cells fromthe appropriate DNA vector. The activity of such ribozymes can beaugmented by their release from the primary transcript by a secondribozyme (Int. Pat. Appl. Publ. No. WO 93/23569, and Int. Pat. Appl.Publ. No. WO 94/02595, both hereby incorporated by reference; Ohkawa etal., 1992; Taira et al., 1991; and Ventura et al., 1993).

Ribozymes may be added directly, or can be complexed with cationiclipids, lipid complexes, packaged within liposomes, or otherwisedelivered to target cells. The RNA or RNA complexes can be locallyadministered to relevant tissues ex vivo, or in vivo through injection,aerosol inhalation, infusion pump or stent, with or without theirincorporation in biopolymers.

Ribozymes may be designed as described in Int. Pat. Appl. Publ. No. WO93/23569 and Int. Pat. Appl. Publ. No. WO 94/02595, each specificallyincorporated herein by reference) and synthesized to be tested in vitroand in vivo, as described. Such ribozymes can also be optimized fordelivery. While specific examples are provided, those in the art willrecognize that equivalent RNA targets in other species can be utilizedwhen necessary.

Hammerhead or hairpin ribozymes may be individually analyzed by computerfolding (Jaeger et al., 1989) to assess whether the ribozyme sequencesfold into the appropriate secondary structure. Those ribozymes withunfavorable intramolecular interactions between the binding arms and thecatalytic core are eliminated from consideration. Varying binding armlengths can be chosen to optimize activity. Generally, at least 5 or sobases on each arm are able to bind to, or otherwise interact with, thetarget RNA.

Ribozymes of the hammerhead or hairpin motif may be designed to annealto various sites in the mRNA message, and can be chemically synthesized.The method of synthesis used follows the procedure for normal RNAsynthesis as described in Usman et al. (1987) and in Scaringe et al.(1990) and makes use of common nucleic acid protecting and couplinggroups, such as dimethoxytrityl at the 5′-end, and phosphoramidites atthe 3′-end. Average stepwise coupling yields are typically >98%. Hairpinribozymes may be synthesized in two parts and annealed to reconstruct anactive ribozyme (Chowrira and Burke, 1992). Ribozymes may be modifiedextensively to enhance stability by modification with nuclease resistantgroups, for example, 2′-amino, 2′-C-allyl, 2′-flouro, 2′-o-methyl, 2′-H(for a review see e.g., Usman and Cedergren, 1992). Ribozymes may bepurified by gel electrophoresis using general methods or by highpressure liquid chromatography and resuspended in water.

Ribozyme activity can be optimized by altering the length of theribozyme binding arms, or chemically synthesizing ribozymes withmodifications that prevent their degradation by serum ribonucleases (seee.g., Int. Pat. Appl. Publ. No. WO 92/07065; Perrault et al, 1990;Pieken et al., 1991; Usman and Cedergren, 1992; Int. Pat. Appl. Publ.No. WO 93/15187; Int. Pat. Appl. Publ. No. WO 91/03162; Eur. Pat. Appl.Publ. No. 92110298.4; U.S. Pat. No. 5,334,711; and Int. Pat. Appl. Publ.No. WO 94/13688, which describe various chemical modifications that canbe made to the sugar moieties of enzymatic RNA molecules), modificationswhich enhance their efficacy in cells, and removal of stem II bases toshorten RNA synthesis times and reduce chemical requirements.

Sullivan et al. (Int. Pat. Appl. Publ. No. WO 94/02595) describes thegeneral methods for delivery of enzymatic RNA molecules. Ribozymes maybe administered to cells by a variety of methods known to those familiarto the art, including, but not restricted to, encapsulation inliposomes, by iontophoresis, or by incorporation into other vehicles,such as hydrogels, cyclodextrins, biodegradable nanocapsules, andbioadhesive microspheres. For some indications, ribozymes may bedirectly delivered ex vivo to cells or tissues with or without theaforementioned vehicles. Alternatively, the RNA/vehicle combination maybe locally delivered by direct inhalation, by direct injection or by useof a catheter, infusion pump or stent. Other routes of delivery include,but are not limited to, intravascular, intramuscular, subcutaneous orjoint injection, aerosol inhalation, oral (tablet or pill form),topical, systemic, ocular, intraperitoneal and/or intrathecal delivery.More detailed descriptions of ribozyme delivery and administration areprovided in Int. Pat. Appl. Publ. No. WO 94/02595 and Int. Pat. Appl.Publ. No. WO 93/23569, each specifically incorporated herein byreference.

Another means of accumulating high concentrations of a ribozyme(s)within cells is to incorporate the ribozyme-encoding sequences into aDNA expression vector. Transcription of the ribozyme sequences aredriven from a promoter for eukaryotic RNA polymerase I (pol I), RNApolymerase II (pol II), or RNA polymerase III (pol III). Transcriptsfrom pol II or pol III promoters will be expressed at high levels in allcells; the levels of a given pol II promoter in a given cell type willdepend on the nature of the gene regulatory sequences (enhancers,silencers, etc.) present nearby. Prokaryotic RNA polymerase promotersmay also be used, providing that the prokaryotic RNA polymerase enzymeis expressed in the appropriate cells (Elroy-Stein and Moss, 1990; Gaoand Huang, 1993; Lieber et al., 1993; Zhou et al., 1990). Ribozymesexpressed from such promoters can function in mammalian cells (e.g.Kashani-Saber et al, 1992; Ojwang et al., 1992; Chen et al., 1992; Yu etal., 1993; L'Huillier et al., 1992; Lisziewicz et al., 1993). Suchtranscription units can be incorporated into a variety of vectors forintroduction into mammalian cells, including but not restricted to,plasmid DNA vectors, viral DNA vectors (such as adenovirus oradeno-associated vectors), or viral RNA vectors (such as retroviral,semliki forest virus, sindbis virus vectors).

Ribozymes may be used as diagnostic tools to examine genetic drift andmutations within diseased cells. They can also be used to assess levelsof the target RNA molecule. The close relationship between ribozymeactivity and the structure of the target RNA allows the detection ofmutations in any region of the molecule which alters the base-pairingand three-dimensional structure of the target RNA. By using multipleribozymes, one may map nucleotide changes which are important to RNAstructure and function in vitro, as well as in cells and tissues.Cleavage of target RNAs with ribozymes may be used to inhibit geneexpression and define the role (essentially) of specified gene productsin the progression of disease. In this manner, other genetic targets maybe defined as important mediators of the disease. These studies willlead to better treatment of the disease progression by affording thepossibility of combinational therapies (e.g., multiple ribozymestargeted to different genes, ribozymes coupled with known small moleculeinhibitors, or intermittent treatment with combinations of ribozymesand/or other chemical or biological molecules). Other in vitro uses ofribozymes are well known in the art, and include detection of thepresence of mRNA associated with an IL-5 related condition. Such RNA isdetected by determining the presence of a cleavage product aftertreatment with a ribozyme using standard methodology.

4.18 Peptide Nucleic Acids

In certain embodiments, the inventors contemplate the use of peptidenucleic acids (PNAs) in the practice of the methods of the invention.PNA is a DNA mimic in which the nucleobases are attached to apseudopeptide backbone (Good and Nielsen, 1997). PNA is able to beutilized in a number methods that traditionally have used RNA or DNA.Often PNA sequences perform better in techniques than the correspondingRNA or DNA sequences and have utilities that are not inherent to RNA orDNA. A review of PNA including methods of making, characteristics of,and methods of using, is provided by Corey (1997) and is incorporatedherein by reference. As such, in certain embodiments, one may preparePNA sequences that are complementary to one or more portions of the ACEmRNA sequence, and such PNA compositions may be used to regulate, alter,decrease, or reduce the translation of ACE-specific mRNA, and therebyalter the level of ACE activity in a host cell to which such PNAcompositions have been administered.

PNAs have 2-aminoethyl-glycine linkages replacing the normalphosphodiester backbone of DNA (Nielsen et al., 1991; Hanvey et al.,1992; Hyrup and Nielsen, 1996; Neilsen, 1996). This chemistry has threeimportant consequences: firstly, in contrast to DNA or phosphorothioateoligonucleotides, PNAs are neutral molecules; secondly, PNAs are achiralwhich avoids the need to develop a stereoselective synthesis; andthirdly, PNA synthesis uses standard Boc (Dueholm et al., 1994) or Fmoc(Thomson et al., 1995) protocols for solid-phase peptide synthesis,although other methods, including a modified Merrifield method, havebeen used (Christensen et al., 1995).

PNA monomers or ready-made oligomers are commercially available fromPerSeptive Biosystems (Framingham, Mass.). PNA syntheses by either Bocor Fmoc protocols are straightforward using manual or automatedprotocols (Norton et al, 1995). The manual protocol lends itself to theproduction of chemically modified PNAs or the simultaneous synthesis offamilies of closely related PNAs.

As with peptide synthesis, the success of a particular PNA synthesiswill depend on the properties of the chosen sequence. For example, whilein theory PNAs can incorporate any combination of nucleotide bases, thepresence of adjacent purines can lead to deletions of one or moreresidues in the product. In expectation of this difficulty, it issuggested that, in producing PNAs with adjacent purines, one shouldrepeat the coupling of residues likely to be added inefficiently. Thisshould be followed by the purification of PNAs by reverse-phasehigh-pressure liquid chromatography (Norton et al., 1995) providingyields and purity of product similar to those observed during thesynthesis of peptides.

Modifications of PNAs for a given application may be accomplished bycoupling amino acids during solid-phase synthesis or by attachingcompounds that contain a carboxylic acid group to the exposed N-terminalamine. Alternatively, PNAs can be modified after synthesis by couplingto an introduced lysine or cysteine. The ease with which PNAs can bemodified facilitates optimization for better solubility or for specificfunctional requirements. Once synthesized, the identity of PNAs andtheir derivatives can be confirmed by mass spectrometry. Several studieshave made and utilized modifications of PNAs (Norton et al., 1995;Haaima et al., 1996; Stetsenko et al., 1996; Petersen et al., 1995;Ulmann et al., 1996; Koch et al., 1995; Orum et al., 1995; Footer etal., 1996; Griffith et al., 1995; Kremsky et al., 1996; Pardridge etal., 1995; Boffa et al., 1995; Landsdorp et al., 1996;Gambacorti-Passerini et al., 1996; Armitage et al., 1997; Seeger et al,1997; Ruskowski et al., 1997). U.S. Pat. No. 5,700,922 discussesPNA-DNA-PNA chimeric molecules and their uses in diagnostics, modulatingprotein in organisms, and treatment of conditions susceptible totherapeutics.

In contrast to DNA and RNA, which contain negatively charged linkages,the PNA backbone is neutral. In spite of this dramatic alteration, PNAsrecognize complementary DNA and RNA by Watson-Crick pairing (Egholm etal., 1993), validating the initial modeling by Nielsen et al. (1991).PNAs lack 3′ to 5′ polarity and can bind in either parallel oranti-parallel fashion, with the anti-parallel mode being preferred(Egholm et al, 1993).

Hybridization of DNA oligonucleotides to DNA and RNA is destabilized byelectrostatic repulsion between the negatively charged phosphatebackbones of the complementary strands. By contrast, the absence ofcharge repulsion in PNA-DNA or PNA-RNA duplexes increases the meltingtemperature (T_(m)) and reduces the dependence of T_(m) on theconcentration of mono- or divalent cations (Nielsen et al., 1991). Theenhanced rate and affinity of hybridization are significant because theyare responsible for the surprising ability of PNAs to perform strandinvasion of complementary sequences within relaxed double-stranded DNA.In addition, the efficient hybridization at inverted repeats suggeststhat PNAs can recognize secondary structure effectively withindouble-stranded DNA. Enhanced recognition also occurs with PNAsimmobilized on surfaces, and Wang et al. have shown that support-boundPNAs can be used to detect hybridization events (Wang et al., 1996).

One might expect that tight binding of PNAs to complementary sequenceswould also increase binding to similar (but not identical) sequences,reducing the sequence specificity of PNA recognition. As with DNAhybridization, however, selective recognition can be achieved bybalancing oligomer length and incubation temperature. Moreover,selective hybridization of PNAs is encouraged by PNA-DNA hybridizationbeing less tolerant of base mismatches than DNA-DNA hybridization. Forexample, a single mismatch within a 16 bp PNA-DNA duplex can reduce theT_(m) by up to 15° C. (Egholm et al., 1993). This high level ofdiscrimination has allowed the development of several PNA-basedstrategies for the analysis of point mutations (Wang et al., 1996;Carlsson et al., 1996; Thiede et al., 1996; Webb and Hurskainen, 1996;Perry-O'Keefe et al., 1996).

High-affinity binding provides clear advantages for molecularrecognition and the development of new applications for PNAs. Forexample, 11-13 nucleotide PNAs inhibit the activity of telomerase, aribonucleo-protein that extends telomere ends using an essential RNAtemplate, while the analogous DNA oligomers do not (Norton et al.,1996).

Neutral PNAs are more hydrophobic than analogous DNA oligomers, and thiscan lead to difficulty solubilizing them at neutral pH, especially ifthe PNAs have a high purine content or if they have the potential toform secondary structures. Their solubility can be enhanced by attachingone or more positive charges to the PNA termini (Nielsen et al., 1991).

Findings by Allfrey and colleagues suggest that strand invasion willoccur spontaneously at sequences within chromosomal DNA (Boffa et al.,1995; Boffa et al., 1996). These studies targeted PNAs to tripletrepeats of the nucleotides CAG and used this recognition to purifytranscriptionally active DNA (Boffa et al., 1995) and to inhibittranscription (Boffa et al., 1996). This result suggests that if PNAscan be delivered within cells then they will have the potential to begeneral sequence-specific regulators of gene expression. Studies andreviews concerning the use of PNAs as antisense and anti-gene agentsinclude Nielsen et al. (1993b), Hanvey et al. (1992), and Good andNielsen (1997). Koppelhus et al. (1997) have used PNAs to inhibit HIV-1inverse transcription, showing that PNAs may be used for antiviraltherapies.

Methods of characterizing the antisense binding properties of PNAs arediscussed in Rose (1993) and Jensen et al. (1997). Rose uses capillarygel electrophoresis to determine binding of PNAs to their complementaryoligonucleotide, measuring the relative binding kinetics andstoichiometry. Similar types of measurements were made by Jensen et al.using BIAcore™ technology.

Other applications of PNAs include use in DNA strand invasion (Nielsenet al., 1991), antisense inhibition (Hanvey et al., 1992), mutationalanalysis (Orum et al., 1993), enhancers of transcription (Mollegaard etal., 1994), nucleic acid purification (Orum et al., 1995), isolation oftranscriptionally active genes (Boffa et al., 1995), blocking oftranscription factor binding (Vickers et al., 1995), genome cleavage(Veselkov et al., 1996), biosensors (Wang et al., 1996), in situhybridization (Thisted et al., 1996), and in a alternative to Southernblotting (Perry-O'Keefe, 1996).

4.19 Polypeptide, Peptides and Peptide Variants

The present invention, in other aspects, provides polypeptidecompositions. Generally, a polypeptide of the invention will be anisolated polypeptide (or an epitope, variant, or active fragmentthereof) derived from a mammalian species. Preferably, the polypeptideis encoded by a polynucleotide sequence disclosed herein or a sequencewhich hybridizes under moderately stringent conditions to apolynucleotide sequence disclosed herein. Alternatively, the polypeptidemay be defined as a polypeptide which comprises a contiguous amino acidsequence from an amino acid sequence disclosed herein, or whichpolypeptide comprises an entire amino acid sequence disclosed herein.

In the present invention, a polypeptide composition is also understoodto comprise one or more polypeptides that are immunologically reactivewith antibodies generated against a polypeptide of the invention,particularly a polypeptide having the amino acid sequence encoded by thedisclosed polynucleotides, or to active fragments, or to variants orbiological functional equivalents thereof.

Likewise, a polypeptide composition of the present invention isunderstood to comprise one or more polypeptides that are capable ofeliciting antibodies that are immunologically reactive with one or morepolypeptides encoded by one or more contiguous nucleic acid sequencesdisclosed in this application, or to active fragments, or to variantsthereof, or to one or more nucleic acid sequences which hybridize to oneor more of these sequences under conditions of moderate to highstringency.

As used herein, an active fragment of a polypeptide includes a whole ora portion of a polypeptide which is modified by conventional techniques,e.g., mutagenesis, or by addition, deletion, or substitution, but whichactive fragment exhibits substantially the same structure function,antigenicity, etc., as a polypeptide as described herein.

In certain illustrative embodiments, the polypeptides of the inventionwill comprise at least an immunogenic portion of a hematologicalmalignancy-related tumor protein or a variant thereof, as describedherein. As noted above, a “hematological malignancy-related tumorprotein” is a protein that is expressed by hematologicalmalignancy-related tumor cells. Proteins that are hematologicalmalignancy-related tumor proteins also react detectably within animmunoassay (such as an ELISA) with antisera from a patient withhematological malignancy. Polypeptides as described herein may be of anylength. Additional sequences derived from the native protein and/orheterologous sequences may be present, and such sequences may (but neednot) possess further immunogenic or antigenic properties.

An “immunogenic portion,” as used herein is a portion of a protein thatis recognized (i.e., specifically bound) by a B-cell and/or T-cellsurface antigen receptor. Such immunogenic portions generally compriseat least 5 amino acid residues, more preferably at least 10, and stillmore preferably at least 20 amino acid residues of a hematologicalmalignancy-related tumor protein or a variant thereof. Certain preferredimmunogenic portions include peptides in which an N-terminal leadersequence and/or transmembrane domain have been deleted. Other preferredimmunogenic portions may contain a small N- and/or C-terminal deletion(e.g., 1-30 amino acids, preferably 5-15 amino acids), relative to themature protein.

Immunogenic portions may generally be identified using well knowntechniques, such as those summarized in Paul, Fundamental Immunology,3rd ed., 243-247 (Raven Press, 1993) and references cited therein. Suchtechniques include screening polypeptides for the ability to react withantigen-specific antibodies, antisera and/or T-cell lines or clones. Asused herein, antisera and antibodies are “antigen-specific” if theyspecifically bind to an antigen (i.e., they react with the protein in anELISA or other immunoassay, and do not react detectably with unrelatedproteins). Such antisera and antibodies may be prepared as describedherein, and using well known techniques. An immunogenic portion of anative hematological malignancy-related tumor protein is a portion thatreacts with such antisera and/or T-cells at a level that is notsubstantially less than the reactivity of the full length polypeptide(e.g., in an ELISA and/or T-cell reactivity assay). Such immunogenicportions may react within such assays at a level that is similar to orgreater than the reactivity of the full length polypeptide. Such screensmay generally be performed using methods well known to those of ordinaryskill in the art, such as those described in Harlow and Lane,Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, 1988.For example, a polypeptide may be immobilized on a solid support andcontacted with patient sera to allow binding of antibodies within thesera to the immobilized polypeptide. Unbound sera may then be removedand bound antibodies detected using, for example, ¹²⁵I-labeled ProteinA.

As noted above, a composition may comprise a variant of a nativehematological malignancy-related tumor protein. A polypeptide “variant,”as used herein, is a polypeptide that differs from a nativehematological malignancy-related tumor protein in one or moresubstitutions, deletions, additions and/or insertions, such that theimmunogenicity of the polypeptide is not substantially diminished. Inother words, the ability of a variant to react with antigen-specificantisera may be enhanced or unchanged, relative to the native protein,or may be diminished by less than 50%, and preferably less than 20%,relative to the native protein. Such variants may generally beidentified by modifying one of the above polypeptide sequences andevaluating the reactivity of the modified polypeptide withantigen-specific antibodies or antisera as described herein. Preferredvariants include those in which one or more portions, such as anN-terminal leader sequence or transmembrane domain, have been removed.Other preferred variants include variants in which a small portion(e.g., 1-30 amino acids, preferably 5-15 amino acids) has been removedfrom the N- and/or C-terminal of the mature protein.

Polypeptide variants encompassed by the present invention include thoseexhibiting at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, or 99% or more identity (determined as describedabove) to the polypeptides disclosed herein.

Preferably, a variant contains conservative substitutions. A“conservative substitution” is one in which an amino acid is substitutedfor another amino acid that has similar properties, such that oneskilled in the art of peptide chemistry would expect the secondarystructure and hydropathic nature of the polypeptide to be substantiallyunchanged. Amino acid substitutions may generally be made on the basisof similarity in polarity, charge, solubility, hydrophobicity,hydrophilicity and/or the amphipathic nature of the residues. Forexample, negatively charged amino acids include aspartic acid andglutamic acid; positively charged amino acids include lysine andarginine; and amino acids with uncharged polar head groups havingsimilar hydrophilicity values include leucine, isoleucine and valine;glycine and alanine; asparagine and glutamine; and serine, threonine,phenylalanine and tyrosine. Other groups of amino acids that mayrepresent conservative changes include: (1) ala, pro, gly, glu, asp,gln, asn, ser, thr, (2) cys, ser, tyr, thr; (3) val, ile, leu, met, ala,phe; (4) lys, arg, his; and (5) phe, tyr, trp, his. A variant may also,or alternatively, contain nonconservative changes. In a preferredembodiment, variant polypeptides differ from a native sequence bysubstitution, deletion or addition of five amino acids or fewer.Variants may also (or alternatively) be modified by, for example, thedeletion or addition of amino acids that have minimal influence on theimmunogenicity, secondary structure and hydropathic nature of thepolypeptide.

As noted above, polypeptides may comprise a signal (or leader) sequenceat the N-terminal end of the protein, which co-translationally orpost-translationally directs transfer of the protein. The polypeptidemay also be conjugated to a linker or other sequence for ease ofsynthesis, purification or identification of the polypeptide (e.g.,poly-His), or to enhance binding of the polypeptide to a solid support.For example, a polypeptide may be conjugated to an immunoglobulin Fcregion.

Polypeptides may be prepared using any of a variety of well knowntechniques. Recombinant polypeptides encoded by DNA sequences asdescribed above may be readily prepared from the DNA sequences using anyof a variety of expression vectors known to those of ordinary skill inthe art. Expression may be achieved in any appropriate host cell thathas been transformed or transfected with an expression vector containinga DNA molecule that encodes a recombinant polypeptide. Suitable hostcells include prokaryotes, yeast, and higher eukaryotic cells, such asmammalian cells and plant cells. Preferably, the host cells employed areE. coli, yeast or a mammalian cell line such as COS or CHO. Supernatantsfrom suitable host/vector systems which secrete recombinant protein orpolypeptide into culture media may be first concentrated using acommercially available filter. Following concentration, the concentratemay be applied to a suitable purification matrix such as an affinitymatrix or an ion exchange resin. Finally, one or more reverse phase HPLCsteps can be employed to further purify a recombinant polypeptide.

Portions and other variants having less than about 100 amino acids, andgenerally less than about 50 amino acids, may also be generated bysynthetic means, using techniques well known to those of ordinary skillin the art. For example, such polypeptides may be synthesized using anyof the commercially available solid-phase techniques, such as theMerrifield solid-phase synthesis method, where amino acids aresequentially added to a growing amino acid chain. See Merrifield, J. Am.Chem. Soc. 85:2149-2146, 1963. Equipment for automated synthesis ofpolypeptides is commercially available from suppliers such as PerkinElmer/Applied BioSystems Division (Foster City, Calif.), and may beoperated according to the manufacturer's instructions.

Within certain specific embodiments, a polypeptide may be a fusionprotein that comprises multiple polypeptides as described herein, orthat comprises at least one polypeptide as described herein and anunrelated sequence, such as a known tumor protein. A fusion partner may,for example, assist in providing T helper epitopes (an immunologicalfusion partner), preferably T helper epitopes recognized by humans, ormay assist in expressing the protein (an expression enhancer) at higheryields than the native recombinant protein. Certain preferred fusionpartners are both immunological and expression enhancing fusionpartners. Other fusion partners may be selected so as to increase thesolubility of the protein or to enable the protein to be targeted todesired intracellular compartments. Still further fusion partnersinclude affinity tags, which facilitate purification of the protein.

Fusion proteins may generally be prepared using standard techniques,including chemical conjugation. Preferably, a fusion protein isexpressed as a recombinant protein, allowing the production of increasedlevels, relative to a non-fused protein, in an expression system.Briefly, DNA sequences encoding the polypeptide components may beassembled separately, and ligated into an appropriate expression vector.The 3′ end of the DNA sequence encoding one polypeptide component isligated, with or without a peptide linker, to the 5′ end of a DNAsequence encoding the second polypeptide component so that the readingframes of the sequences are in phase. This permits translation into asingle fusion protein that retains the biological activity of bothcomponent polypeptides.

A peptide linker sequence may be employed to separate the first andsecond polypeptide components by a distance sufficient to ensure thateach polypeptide folds into its secondary and tertiary structures. Sucha peptide linker sequence is incorporated into the fusion protein usingstandard techniques well known in the art. Suitable peptide linkersequences may be chosen based on the following factors: (1) theirability to adopt a flexible extended conformation; (2) their inabilityto adopt a secondary structure that could interact with functionalepitopes on the first and second polypeptides; and (3) the lack ofhydrophobic or charged residues that might react with the polypeptidefunctional epitopes. Preferred peptide linker sequences contain Gly, Asnand Ser residues. Other near neutral amino acids, such as Thr and Alamay also be used in the linker sequence. Amino acid sequences which maybe usefully employed as linkers include those disclosed in Maratea etal., Gene 40:39-46, 1985; Murphy et al., Proc. Natl. Acad. Sci. USA83:8258-8262, 1986; U.S. Pat. No. 4,935,233 and U.S. Pat. No. 4,751,180.The linker sequence may generally be from 1 to about 50 amino acids inlength. Linker sequences are not required when the first and secondpolypeptides have non-essential N-terminal amino acid regions that canbe used to separate the functional domains and prevent stericinterference.

The ligated DNA sequences are operably linked to suitabletranscriptional or translational regulatory elements. The regulatoryelements responsible for expression of DNA are located only 5′ to theDNA sequence encoding the first polypeptides. Similarly, stop codonsrequired to end translation and transcription termination signals areonly present 3′ to the DNA sequence encoding the second polypeptide.

Fusion proteins are also provided. Such proteins comprise a polypeptideas described herein together with an unrelated immunogenic protein.Preferably the immunogenic protein is capable of eliciting a recallresponse. Examples of such proteins include tetanus, tuberculosis andhepatitis proteins (see, for example, Stoute et al. New Engl. J. Med.,336:86-91, 1997).

Within preferred embodiments, an immunological fusion partner is derivedfrom protein D, a surface protein of the gram-negative bacteriumHaemophilus influenza B (WO 91/18926). Preferably, a protein Dderivative comprises approximately the first third of the protein (e.g.,the first N-terminal 100-110 amino acids), and a protein D derivativemay be lipidated. Within certain preferred embodiments, the first 109residues of a Lipoprotein D fusion partner is included on the N-terminusto provide the polypeptide with additional exogenous T-cell epitopes andto increase the expression level in E. coli (thus functioning as anexpression enhancer). The lipid tail ensures optimal presentation of theantigen to antigen presenting cells. Other fusion partners include thenon-structural protein from influenzae virus, NS1 (hemagglutinin).Typically, the N-terminal 81 amino acids are used, although differentfragments that include T-helper epitopes may be used.

In another embodiment, the immunological fusion partner is the proteinknown as LYTA, or a portion thereof (preferably a C-terminal portion).LYTA is derived from Streptococcus pneumoniae, which synthesizes anN-acetyl-L-alanine amidase known as amidase LYTA (encoded by the LytAgene; Gene 43:265-292, 1986). LYTA is an autolysin that specificallydegrades certain bonds in the peptidoglycan backbone. The C-terminaldomain of the LYTA protein is responsible for the affinity to thecholine or to some choline analogues such as DEAE. This property hasbeen exploited for the development of E. coli C-LYTA expressing plasmidsuseful for expression of fusion proteins. Purification of hybridproteins containing the C-LYTA fragment at the amino terminus has beendescribed (see Biotechnology 10:795-798, 1992). Within a preferredembodiment, a repeat portion of LYTA may be incorporated into a fusionprotein. A repeat portion is found in the C-terminal region starting atresidue 178. A particularly preferred repeat portion incorporatesresidues 188-305.

In general, polypeptides (including fusion proteins) and polynucleotidesas described herein are isolated. An “isolated” polypeptide orpolynucleotide is one that is removed from its original environment Forexample, a naturally-occurring protein is isolated if it is separatedfrom some or all of the coexisting materials in the natural system.Preferably, such polypeptides are at least about 90% pure, morepreferably at least about 95% pure and most preferably at least about99% pure. A polynucleotide is considered to be isolated if, for example,it is cloned into a vector that is not a part of the naturalenvironment.

4.20 Binding Agents

The present invention further employs agents, such as antibodies andantigen-binding fragments thereof, that specifically bind to ahematological malignancy-related antigen. As used herein, an antibody,or antigen-binding fragment thereof, is said to “specifically bind” to ahematological malignancy-related antigen if it reacts at a detectablelevel (within, for example, an ELISA) with, and does not reactdetectably with unrelated proteins under similar conditions. As usedherein, “binding” refers to a noncovalent association between twoseparate molecules such that a complex is formed. The ability to bindmay be evaluated by, for example, determining a binding constant for theformation of the complex. The binding constant is the value obtainedwhen the concentration of the complex is divided by the product of thecomponent concentrations. In general, two compounds are said to “bind,”in the context of the present invention, when the binding constant forcomplex formation exceeds about 10³ L/mol. The binding constant maybedetermined using methods well known in the art.

Binding agents may be further capable of differentiating betweenpatients with and without a hematological malignancy. Such bindingagents generate a signal indicating the presence of a hematologicalmalignancy in at least about 20% of patients with the disease, and willgenerate a negative signal indicating the absence of the disease in atleast about 90% of individuals without the disease. To determine whethera binding agent satisfies this requirement, biological samples (e.g.,blood, sera, urine and/or tumor biopsies) from patients with and withouta hematological malignancy (as determined using standard clinical tests)may be assayed as described herein for the presence of polypeptides thatbind to the binding agent. It will be apparent that a statisticallysignificant number of samples with and without the disease should beassayed. Each binding agent should satisfy the above criteria; however,those of ordinary skill in the art will recognize that binding agentsmay be used in combination to improve sensitivity.

Any agent that satisfies the above requirements may be a binding agent.For example, a binding agent may be a ribosome, with or without apeptide component, an RNA molecule or a polypeptide. In a preferredembodiment, a binding agent is an antibody or an antigen-bindingfragment thereof. Antibodies may be prepared by any of a variety oftechniques known to those of ordinary skill in the art. See, e.g.,Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring HarborLaboratory, 1988. In general, antibodies can be produced by cell culturetechniques, including the generation of monoclonal antibodies asdescribed herein, or via transfection of antibody genes into suitablebacterial or mammalian cell hosts, in order to allow for the productionof recombinant antibodies. In one technique, an immunogen comprising thepolypeptide is initially injected into any of a wide variety of mammals(e.g., mice, rats, rabbits, sheep or goats). In this step, thepolypeptides of this invention may serve as the immunogen withoutmodification. Alternatively, particularly for relatively shortpolypeptides, a superior immune response may be elicited if thepolypeptide is joined to a carrier protein, such as bovine serum albuminor keyhole limpet hemocyanin. The immunogen is injected into the animalhost, preferably according to a predetermined schedule incorporating oneor more booster immunizations, and the animals are bled periodically.Polyclonal antibodies specific for the polypeptide may then be purifiedfrom such antisera by, for example, affinity chromatography using thepolypeptide coupled to a suitable solid support.

Monoclonal antibodies specific for an antigenic polypeptide of interestmay be prepared, for example, using the technique of Kohler andMilstein, Eur. J. Immunol. 6:511-519, 1976, and improvements thereto.Briefly, these methods involve the preparation of immortal cell linescapable of producing antibodies having the desired specificity (i.e.,reactivity with the polypeptide of interest). Such cell lines may beproduced, for example, from spleen cells obtained from an animalimmunized as described above. The spleen cells are then immortalized by,for example, fusion with a myeloma cell fusion partner, preferably onethat is syngeneic with the immunized animal. A variety of fusiontechniques may be employed. For example, the spleen cells and myelomacells may be combined with a nonionic detergent for a few minutes andthen plated at low density on a selective medium that supports thegrowth of hybrid cells, but not myeloma cells. A preferred selectiontechnique uses HAT (hypoxanthine, aminopterin, thymidine) selection.After a sufficient time, usually about 1 to 2 weeks, colonies of hybridsare observed. Single colonies are selected and their culturesupernatants tested for binding activity against the polypeptide.Hybridomas having high reactivity and specificity are preferred.

Monoclonal antibodies may be isolated from the supernatants of growinghybridoma colonies. In addition, various techniques may be employed toenhance the yield, such as injection of the hybridoma cell line into theperitoneal cavity of a suitable vertebrate host, such as a mouse.Monoclonal antibodies may then be harvested from the ascites fluid orthe blood. Contaminants may be removed from the antibodies byconventional techniques, such as chromatography, gel filtration,precipitation, and extraction. The polypeptides of this invention may beused in the purification process in, for example, an affinitychromatography step.

Within certain embodiments, the use of antigen-binding fragments ofantibodies may be preferred. Such fragments include Fab fragments, whichmay be prepared using standard techniques. Briefly, immunoglobulins maybe purified from rabbit serum by affinity chromatography on Protein Abead columns (Harlow and Lane, Antibodies: A Laboratory Manual, ColdSpring Harbor Laboratory, 1988) and digested by papain to yield Fab andFc fragments. The Fab and Fc fragments may be separated by affinitychromatography on protein A bead columns.

Monoclonal antibodies, and fragments thereof, of the present inventionmay be coupled to one or more therapeutic agents, such as radionuclides,differentiation inducers, drugs, toxins, and derivatives thereof.Preferred radionuclides include ⁹⁰Y, ¹²³I, ¹²⁵I, ¹³¹I, ¹⁸⁶Re, ¹⁸⁸Re,²¹¹At, and ²¹²Bi. Preferred drugs include methotrexate, and pyrimidineand purine analogs. Preferred differentiation inducers include phorbolesters and butyric acid. Preferred toxins include ricin, abrin,diptheria toxin, cholera toxin, gelonin, Pseudomonas exotoxin, Shigellatoxin, and pokeweed antiviral protein. For certain in vivo and ex vivotherapies, an antibody or fragment thereof is preferably coupled to acytotoxic agent, such as a radioactive or chemotherapeutic moiety.

A therapeutic agent may be coupled (e.g., covalently bonded) to asuitable monoclonal antibody either directly or indirectly (e.g., via alinker group). A direct reaction between an agent and an antibody ispossible when each possesses a substituent capable of reacting with theother. For example, a nucleophilic group, such as an amino or sulfhydrylgroup, on one may be capable of reacting with a carbonyl-containinggroup, such as an anhydride an acid halide, or with an alkyl groupcontaining a good leaving group (e.g., a halide) on the other.

Alternatively, it may be desirable to couple a therapeutic agent and anantibody via a linker group. A linker group can function as a spacer todistance an antibody from an agent in order to avoid interference withbinding capabilities. A linker group can also serve to increase thechemical reactivity of a substituent on an agent or an antibody, andthus increase the coupling efficiency. An increase in chemicalreactivity may also facilitate the use of agents, or functional groupson agents, which otherwise would not be possible.

It will be evident to those skilled in the art that a variety ofbifunctional or polyfunctional reagents, both homo- andhetero-functional (such as those described in the catalog of the PierceChemical Co., Rockford, Ill.), may be employed as the linker group.Coupling may be effected, for example, through amino groups, carboxylgroups, sulfhydryl groups or oxidized carbohydrate residues. There arenumerous references describing such methodology, e.g., U.S. Pat. No.4,671,958.

Where a therapeutic agent is more potent when free from the antibodyportion of the immunoconjugates of the present invention, it may bedesirable to use a linker group which is cleavable during or uponinternalization into a cell. A number of different cleavable linkergroups have been described. The mechanisms for the intracellular releaseof an agent from these linker groups include cleavage by reduction of adisulfide bond (e.g., U.S. Pat. No. 4,489,710), by irradiation of aphotolabile bond (e.g., U.S. Pat. No. 4,625,014), by hydrolysis ofderivatized amino acid side chains (e.g., U.S. Pat. No. 4,638,045), byserum complement-mediated hydrolysis (e.g., U.S. Pat. No. 4,671,958),and acid-catalyzed hydrolysis (e.g., U.S. Pat. No. 4,569,789).

It may be desirable to couple more than one agent to an antibody. In oneembodiment, multiple molecules of an agent are coupled to one antibodymolecule. In another embodiment, more than one type of agent may becoupled to one antibody. Regardless of the particular embodiment,immunoconjugates with more than one agent may be prepared in a varietyof ways. For example, more than one agent may be coupled directly to anantibody molecule, or linkers which provide multiple sites forattachment can be used. Alternatively, a carrier can be used.

A carrier may bear the agents in a variety of ways, including covalentbonding either directly or via a linker group. Suitable carriers includeproteins such as albumins (e.g., U.S. Pat. No. 4,507,234), peptides andpolysaccharides such as aminodextran (e.g. U.S. Pat. No. 4,699,784). Acarrier may also bear an agent by noncovalent bonding or byencapsulation, such as within a liposome vesicle (e.g., U.S. Pat. Nos.4,429,008 and 4,873,088). Carriers specific for radionuclide agentsinclude radiohalogenated small molecules and chelating compounds. Forexample, U.S. Pat. No. 4,735,792 discloses representativeradiohalogenated small molecules and their synthesis. A radionuclidechelate may be formed from chelating compounds that include thosecontaining nitrogen and sulfur atoms as the donor atoms for binding themetal, or metal oxide, radionuclide. For example, U.S. Pat. No.4,673,562 discloses representative chelating compounds and theirsynthesis.

A variety of routes of administration for the antibodies andimmunoconjugates may be used. Typically, administration will beintravenous, intramuscular, subcutaneous or in the bed of a resectedtumor. It will be evident that the precise dose of theantibody/immunoconjugate will vary depending upon the antibody used, theantigen density on the tumor, and the rate of clearance of the antibody.

4.21 Vaccines

In certain preferred embodiments of the present invention, vaccines areprovided. The vaccines will generally comprise one or morepharmaceutical compositions, such as those discussed above, incombination with an immunostimulant. An immunostimulant may be anysubstance that enhances or potentiates an immune response (antibodyand/or cell-mediated) to an exogenous antigen. Examples ofimmunostimulants include adjuvants, biodegradable microspheres (e.g.,polylactic galactide) and liposomes (into which the compound isincorporated; see e.g., Fullerton, U.S. Pat. No. 4,235,877). Vaccinepreparation is generally described in, for example, M. F. Powell and M.J. Newman, eds., “Vaccine Design (the subunit and adjuvant approach),”Plenum Press (NY, 1995). Pharmaceutical compositions and vaccines withinthe scope of the present invention may also contain other compounds,which may be biologically active or inactive. For example, one or moreimmunogenic portions of other tumor antigens may be present, eitherincorporated into a fusion polypeptide or as a separate compound, withinthe composition or vaccine.

Illustrative vaccines may contain DNA encoding one or more of thepolypeptides as described above, such that the polypeptide is generatedin situ. As noted above, the DNA may be present within any of a varietyof delivery systems known to those of ordinary skill in the art,including nucleic acid expression systems, bacteria and viral expressionsystems. Numerous gene delivery techniques are well known in the art,such as those described by Rolland, Crit. Rev. Therap. Drug CarrierSystems 15:143-198, 1998, and references cited therein. Appropriatenucleic acid expression systems contain the necessary DNA sequences forexpression in the patient (such as a suitable promoter and terminatingsignal). Bacterial delivery systems involve the administration of abacterium (such as Bacillus-Calmette-Guerrin) that expresses animmunogenic portion of the polypeptide on its cell surface or secretessuch an epitope. In a preferred embodiment, the DNA may be introducedusing a viral expression system (e.g., vaccinia or other pox virus,retrovirus, or adenovirus), which may involve the use of anon-pathogenic (defective), replication competent virus. Suitablesystems are disclosed, for example, in Fisher-Hoch et al., Proc. Natl.Acad. Sci. USA 86:317-321, 1989; Flexner et al., Ann. N.Y. Acad. Sci.569:86-103, 1989; Flexner et al., Vaccine 8:17-21, 1990; U.S. Pat. Nos.4,603,112, 4,769,330, and 5,017,487; WO 89/01973; U.S. Pat. No.4,777,127; GB 2,200,651; EP 0,345,242; WO 91/02805; Berkner,Biotechniques 6:616-627, 1988; Rosenfeld et al., Science 252:431-434,1991; Kolls et al., Proc. Natl. Acad. Sci. USA 91:215-219, 1994;Kass-Eisler et al., Proc. Natl. Acad. Sci. USA 90:11498-11502, 1993;Guzman et al., Circulation 88:2838-2848, 1993; and Guzman et al., Cir.Res. 73:1202-1207, 1993. Techniques for incorporating DNA into suchexpression systems are well known to those of ordinary skill in the art.The DNA may also be “naked,” as described, for example, in Ulmer et al.,Science 259:1745-1749, 1993 and reviewed by Cohen, Science259:1691-1692, 1993. The uptake of naked DNA may be increased by coatingthe DNA onto biodegradable beads, which are efficiently transported intothe cells. It will be apparent that a vaccine may comprise both apolynucleotide and a polypeptide component. Such vaccines may providefor an enhanced immune response.

It will be apparent that a vaccine may contain pharmaceuticallyacceptable salts of the polynucleotides and polypeptides providedherein. Such salts may be prepared from pharmaceutically acceptablenon-toxic bases, including organic bases (e.g., salts of primary,secondary and tertiary amines and basic amino acids) and inorganic bases(e.g. sodium, potassium, lithium, ammonium, calcium and magnesiumsalts).

While any suitable carrier known to those of ordinary skill in the artmay be employed in the vaccine compositions of this invention, the typeof carrier will vary depending on the mode of administration.Compositions of the present invention may be formulated for anyappropriate manner of administration, including for example, topical,oral, nasal, intravenous, intracranial, intraperitoneal, subcutaneous orintramuscular administration. For parenteral administration, such assubcutaneous injection, the carrier preferably comprises water, saline,alcohol, a fat, a wax or a buffer. For oral administration, any of theabove carriers or a solid carrier, such as mannitol, lactose, starch,magnesium stearate, sodium saccharine, talcum, cellulose, glucose,sucrose, and magnesium carbonate, may be employed. Biodegradablemicrospheres (e.g., polylactate polyglycolate) may also be employed ascarriers for the pharmaceutical compositions of this invention Suitablebiodegradable microspheres are disclosed, for example, in U.S. Pat. Nos.4,897,268; 5,075,109; 5,928,647; 5,811,128; 5,820,883; 5,853,763;5,814,344 and 5,942,252. One may also employ a carrier comprising theparticulate-protein complexes described in U.S. Pat. No. 5,928,647,which are capable of inducing a class I-restricted cytotoxic Tlymphocyte responses in a host.

Such compositions may also comprise buffers (e.g., neutral bufferedsaline or phosphate buffered saline), carbohydrates (e.g., glucose,mannose, sucrose or dextrans), mannitol, proteins, polypeptides or aminoacids such as glycine, antioxidants, bacteriostats, chelating agentssuch as EDTA or glutathione, adjuvants (e.g., aluminum hydroxide),solutes that render the formulation isotonic, hypotonic or weaklyhypertonic with the blood of a recipient, suspending agents, thickeningagents and/or preservatives. Alternatively, compositions of the presentinvention may be formulated as a lyophilizate. Compounds may also beencapsulated within liposomes using well known technology.

Any of a variety of immunostimulants may be employed in the vaccines ofthis invention. For example, an adjuvant may be included. Most adjuvantscontain a substance designed to protect the antigen from rapidcatabolism, such as aluminum hydroxide or mineral oil, and a stimulatorof immune responses, such as lipid A, Bortadella pertussis orMycobacterium tuberculosis derived proteins. Suitable adjuvants arecommercially available as, for example, Freund's Incomplete Adjuvant andComplete Adjuvant (Difco Laboratories, Detroit, Mich.); Merck Adjuvant65 (Merck and Company, Inc., Rahway, N.J.); AS-2 (SmithKline Beecham,Philadelphia, Pa.); aluminum salts such as aluminum hydroxide gel (alum)or aluminum phosphate; salts of calcium, iron or zinc; an insolublesuspension of acylated tyrosine; acylated sugars; cationically oranionically derivatized polysaccharides; polyphosphazenes; biodegradablemicrospheres; monophosphoryl lipid A and quil A. Cytokines, such asGM-CSF or interleukin-2, -7, or -12, may also be used as adjuvants.

Within the vaccines provided herein, the adjuvant composition ispreferably designed to induce an immune response predominantly of theTh1 type. High levels of Th1-type cytokines (e.g., IFN-γ, TNFα, IL-2 andIL-12) tend to favor the induction of cell mediated immune responses toan administered antigen. In contrast, high levels of Th2-type cytokines(e.g., IL-4, IL-5, IL-6 and IL-10) tend to favor the induction ofhumoral immune responses. Following application of a vaccine as providedherein, a patient will support an immune response that includes Th1- andTh2-type responses. Within a preferred embodiment, in which a responseis predominantly Th1-type, the level of Th1-type cytokines will increaseto a greater extent than the level of Th2-type cytokines. The levels ofthese cytokines may be readily assessed using standard assays. For areview of the families of cytokines, see Mosmann and Coffman, Ann. Rev.Immunol. 7:145-173, 1989.

Preferred adjuvants for use in eliciting a predominantly Th1-typeresponse include, for example, a combination of monophosphoryl lipid A,preferably 3-de-O-acylated monophosphoryl lipid A (3D-MPL), togetherwith an aluminum salt. MPL adjuvants are available from CorixaCorporation (Seattle, Wash.; see U.S. Pat. Nos. 4,436,727; 4,877,611;4,866,034 and 4,912,094). CpG-containing oligonucleotides (in which theCpG dinucleotide is unmethylated) also induce a predominantly Th1response. Such oligonucleotides are well known and are described, forexample, in WO 96/02555, WO 99/33488 and U.S. Pat. Nos. 6,008,200 and5,856,462. Immunostimulatory DNA sequences are also described, forexample, by Sato et al., Science 273:352, 1996. Another preferredadjuvant is a saponin, preferably QS21 (Aquila Biopharmaceuticals Inc.,Framingham, Mass.), which may be used alone or in combination with otheradjuvants. For example, an enhanced system involves the combination of amonophosphoryl lipid A and saponin derivative, such as the combinationof QS21 and 3D-MPL as described in WO 94/00153, or a less reactogeniccomposition where the QS21 is quenched with cholesterol, as described inWO 96/33739. Other preferred formulations comprise an oil-in-wateremulsion and tocopherol. A particularly potent adjuvant formulationinvolving QS21, 3D-MPL and tocopherol in an oil-in-water emulsion isdescribed in WO 95/17210.

Other preferred adjuvants include Montanide ISA 720 (Seppic, France),SAF (Chiron, California, United States), ISCOMS (CSL), MF-59 (Chiron),the SBAS series of adjuvants (e.g., SBAS-2 or SBAS-4, available fromSmithKline Beecham, Rixensart, Belgium), Detox (Corixa, Hamilton,Mont.), RC-529 (Corixa, Hamilton, Mont.) and other aminoalkylglucosaminide 4-phosphates (AGPs), such as those described in pendingU.S. patent application Ser. Nos. 08/853,826 and 09/074,720, thedisclosures of which are incorporated herein by reference in theirentireties.

Any vaccine provided herein may be prepared using well known methodsthat result in a combination of antigen, immune response enhancer and asuitable carrier or excipient. The compositions described herein may beadministered as part of a sustained release formulation (i.e., aformulation such as a capsule, sponge or gel (composed ofpolysaccharides, for example) that effects a slow release of compoundfollowing administration). Such formulations may generally be preparedusing well known technology (see, e.g., Coombes et al., Vaccine14:1429-1438, 1996) and administered by, for example, oral, rectal orsubcutaneous implantation, or by implantation at the desired targetsite. Sustained-release formulations may contain a polypeptide,polynucleotide or antibody dispersed in a carrier matrix and/orcontained within a reservoir surrounded by a rate controlling membrane.

Carriers for use within such formulations are biocompatible, and mayalso be biodegradable; preferably the formulation provides a relativelyconstant level of active component release. Such carriers includemicroparticles of poly(lactide-co-glycolide), polyacrylate, latex,starch, cellulose, dextran and the like. Other delayed-release carriersinclude supramolecular biovectors, which comprise a non-liquidhydrophilic core (e.g., a cross-linked polysaccharide oroligosaccharide) and, optionally, an external layer comprising anamphiphilic compound, such as a phospholipid (see e.g., U.S. Pat. No.5,151,254 and PCT applications WO 94/20078, WO/94/23701 and WO96/06638). The amount of active compound contained within a sustainedrelease formulation depends upon the site of implantation, the rate andexpected duration of release and the nature of the condition to betreated or prevented.

Any of a variety of delivery vehicles may be employed withinpharmaceutical compositions and vaccines to facilitate production of anantigen-specific immune response that targets tumor cells. Deliveryvehicles include antigen presenting cells (APCs), such as dendriticcells, macrophages, B cells, monocytes and other cells that may beengineered to be efficient APCs. Such cells may, but need not, begenetically modified to increase the capacity for presenting theantigen, to improve activation and/or maintenance of the T cellresponse, to have anti-tumor effects per se and/or to be immunologicallycompatible with the receiver (i.e., matched HLA haplotype). APCs maygenerally be isolated from any of a variety of biological fluids andorgans, including tumor and peritumoral tissues, and may be autologous,allogeneic, syngeneic or xenogeneic cells.

Certain preferred embodiments of the present invention use dendriticcells or progenitors thereof as antigen-presenting cells. Dendriticcells are highly potent APCs (Banchereau and Steinman, Nature392:245-251, 1998) and have been shown to be effective as aphysiological adjuvant for eliciting prophylactic or therapeuticantitumor immunity (see Timmerman and Levy, Ann. Rev. Med. 50:507-529,1999). In general, dendritic cells may be identified based on theirtypical shape (stellate in situ, with marked cytoplasmic processes(dendrites) visible in vitro), their ability to take up, process andpresent antigens with high efficiency and their ability to activatenaïve T cell responses. Dendritic cells may, of course, be engineered toexpress specific cell-surface receptors or ligands that are not commonlyfound on dendritic cells in vivo or ex vivo, and such modified dendriticcells are contemplated by the present invention. As an alternative todendritic cells, secreted vesicles antigen-loaded dendritic cells(called exosomes) may be used within a vaccine (see Zitvogel et al.,Nature Med. 4:594-600, 1998).

Dendritic cells and progenitors may be obtained from peripheral blood,bone marrow, tumor-infiltrating cells, peritumoral tissues-infiltratingcells, lymph nodes, spleen, skin, umbilical cord blood or any othersuitable tissue or fluid. For example, dendritic cells may bedifferentiated ex vivo by adding a combination of cytokines such asGM-CSF, IL-4, IL-13 and/or TNFα to cultures of monocytes harvested fromperipheral blood. Alternatively, CD34 positive cells harvested fromperipheral blood, umbilical cord blood or bone marrow may bedifferentiated into dendritic cells by adding to the culture mediumcombinations of GM-CSF, IL-3, TNFα, CD40 ligand, LPS, flt3 ligand and/orother compound(s) that induce differentiation, maturation andproliferation of dendritic cells.

Dendritic cells are conveniently categorized as “immature” and “mature”cells, which allows a simple way to discriminate between two wellcharacterized phenotypes. However, this nomenclature should not beconstrued to exclude all possible intermediate stages ofdifferentiation. Immature dendritic cells are characterized as APC witha high capacity for antigen uptake and processing, which correlates withthe high expression of Fcγ receptor and mannose receptor. The maturephenotype is typically characterized by a lower expression of thesemarkers, but a high expression of cell surface molecules responsible forT cell activation such as class I and class II MHC, adhesion molecules(e.g., CD54 and CD11) and costimulatory molecules (e.g., CD40, CD80,CD86 and 4-1BB).

APCs may generally be transfected with a polynucleotide encoding ahematological malignancy-related tumor protein (or portion or othervariant thereof) such that the hematological malignancy-related tumorpolypeptide, or an immunogenic portion thereof, is expressed on the cellsurface. Such transfection may take place ex vivo, and a composition orvaccine comprising such transfected cells may then be used fortherapeutic purposes, as described herein. Alternatively, a genedelivery vehicle that targets a dendritic or other antigen presentingcell may be administered to a patient, resulting in transfection thatoccurs in vivo. In vivo and ex vivo transfection of dendritic cells, forexample, may generally be performed using any methods known in the art,such as those described in WO 97/24447, or the gene gun approachdescribed by Mahvi et al., Immunology and cell Biology 75:456-460, 1997.Antigen loading of dendritic cells may be achieved by incubatingdendritic cells or progenitor cells with the hematologicalmalignancy-related tumor polypeptide, DNA (naked or within a plasmidvector) or RNA; or with antigen-expressing recombinant bacterium orviruses (e.g., vaccinia, fowlpox, adenovirus or lentivirus vectors).Prior to loading, the polypeptide may be covalently conjugated to animmunological partner that provides T cell help (e.g., a carriermolecule). Alternatively, a dendritic cell may be pulsed with anon-conjugated immunological partner, separately or in the presence ofthe polypeptide.

Vaccines and pharmaceutical compositions may be presented in unit-doseor multi-dose containers, such as sealed ampoules or vials. Suchcontainers are preferably hermetically sealed to preserve sterility ofthe formulation until use. In general, formulations may be stored assuspensions, solutions or emulsions in oily or aqueous vehicles.Alternatively, a vaccine or pharmaceutical composition may be stored ina freeze-dried condition requiring only the addition of a sterile liquidcarrier immediately prior to use.

4.22 Cancer Therapy

In further aspects of the present invention, the compositions describedherein may be used for immunotherapy of cancer, such as hematologicalmalignancy. Within such methods, pharmaceutical compositions andvaccines are typically administered to a patient. As used herein, a“patient” refers to any warm-blooded animal, preferably a human. Apatient may or may not be afflicted with cancer. Accordingly, the abovepharmaceutical compositions and vaccines may be used to prevent thedevelopment of a cancer or to treat a patient afflicted with a cancer. Acancer may be diagnosed using criteria generally accepted in the art,including the presence of a malignant tumor. Pharmaceutical compositionsand vaccines may be administered either prior to or following surgicalremoval of primary tumors and/or treatment such as administration ofradiotherapy or conventional chemotherapeutic drugs. Administration maybe by any suitable method, including administration by intravenous,intraperitoneal, intramuscular, subcutaneous, intranasal, intradermal,anal, vaginal, topical and oral routes.

Within certain embodiments, immunotherapy may be active immunotherapy,in which treatment relies on the in vivo stimulation of the endogenoushost immune system to react against tumors with the administration ofimmune response-modifying agents (such as polypeptides andpolynucleotides as provided herein).

Within other embodiments, immunotherapy may be passive immunotherapy, inwhich treatment involves the delivery of agents with establishedtumor-immune reactivity (such as effector cells or antibodies) that candirectly or indirectly mediate antitumor effects and does notnecessarily depend on an intact host immune system. Examples of effectorcells include T cells as discussed above, T lymphocytes (such as CD8⁺cytotoxic T lymphocytes and CD4⁺ T-helper tumor-infiltratinglymphocyte), killer cells (such as Natural Killer cells andlymphokine-activated killer cells), B cells and antigen-presenting cells(such as dendritic cells and macrophages) expressing a polypeptideprovided herein. T cell receptors and antibody receptors specific forthe polypeptides recited herein may be cloned, expressed and transferredinto other vectors or effector cells for adoptive immunotherapy. Thepolypeptides provided herein may also be used to generate antibodies oranti-idiotypic antibodies (as described above and in U.S. Pat. No.4,918,164) for passive immunotherapy.

Effector cells may generally be obtained in sufficient quantities foradoptive immunotherapy by growth in vitro, as described herein. Cultureconditions for expanding single antigen-specific effector cells toseveral billion in number with retention of antigen recognition in vivoare well known in the art. Such in vitro culture conditions typicallyuse intermittent stimulation with antigen, often in the presence ofcytokines (such as IL-2) and non-dividing feeder cells. As noted above,immunoreactive polypeptides as provided herein may be used to rapidlyexpand antigen-specific T cell cultures in order to generate asufficient number of cells for immunotherapy. In particular,antigen-presenting cells, such as dendritic, macrophage, monocyte,fibroblast and/or B cells, may be pulsed with immunoreactivepolypeptides or transfected with one or more polynucleotides usingstandard techniques well known in the art. For example,antigen-presenting cells can be transfected with a polynucleotide havinga promoter appropriate for increasing expression in a recombinant virusor other expression system. Cultured effector cells for use in therapymust be able to grow and distribute widely, and to survive long term invivo. Studies have shown that cultured effector cells can be induced togrow in vivo and to survive long term in substantial numbers by repeatedstimulation with antigen supplemented with IL-2 (see, for example,Cheever et al., Immunological Reviews 157:177, 1997).

Alternatively, a vector expressing a polypeptide recited herein may beintroduced into antigen presenting cells taken from a patient andclonally propagated ex vivo for transplant back into the same patient.Transfected cells may be reintroduced into the patient using any meansknown in the art, preferably in sterile form by intravenous,intracavitary, intraperitoneal or intratumor administration.

Routes and frequency of administration of the therapeutic compositionsdescribed herein, as well as dosage, will vary from individual toindividual, and may be readily established using standard techniques. Ingeneral, the pharmaceutical compositions and vaccines may beadministered by injection (e.g., intracutaneous, intramuscular,intravenous or subcutaneous), intranasally (e.g., by aspiration) ororally. Preferably, between 1 and 10 doses may be administered over a 52week period. Preferably, 6 doses are administered, at intervals of 1month, and booster vaccinations may be given periodically thereafter.Alternate protocols may be appropriate for individual patients. Asuitable dose is an amount of a compound that, when administered asdescribed above, is capable of promoting an anti-tumor immune response,and is at least 10-50% above the basal (i.e., untreated) level. Suchresponse can be monitored by measuring the anti-tumor antibodies in apatient or by vaccine-dependent generation of cytolytic effector cellscapable of killing the patient's tumor cells in vitro. Such vaccinesshould also be capable of causing an immune response that leads to animproved clinical outcome (e.g., more frequent remissions, complete orpartial or longer disease-free survival) in vaccinated patients ascompared to non-vaccinated patients. In general, for pharmaceuticalcompositions and vaccines comprising one or more polypeptides, theamount of each polypeptide present in a dose ranges from about 25 μg to5 mg per kg of host. Suitable dose sizes will vary with the size of thepatient, but will typically range from about 0.1 mL to about 5 mL.

In general, an appropriate dosage and treatment regimen provides theactive compound(s) in an amount sufficient to provide therapeutic and/orprophylactic benefit. Such a response can be monitored by establishingan improved clinical outcome (e.g., more frequent remissions, completeor partial, or longer disease-free survival) in treated patients ascompared to non-treated patients. Increases in preexisting immuneresponses to a hematological malignancy-related tumor protein generallycorrelate with an improved clinical outcome. Such immune responses maygenerally be evaluated using standard proliferation, cytotoxicity orcytokine assays, which may be performed using samples obtained from apatient before and after treatment.

4.23 Cancer Detection and Diagnosis

In general, a cancer may be detected in a patient based on the presenceof one or more hematological malignancy-related tumor proteins and/orpolynucleotides encoding such proteins in a biological sample (forexample, blood, sera, sputum urine and/or tumor biopsies) obtained fromthe patient. In other words, such proteins may be used as markers toindicate the presence or absence of a cancer such as hematologicalmalignancy. In addition, such proteins may be useful for the detectionof other cancers. The binding agents provided herein generally permitdetection of the level of antigen that binds to the agent in thebiological sample. Polynucleotide primers and probes may be used todetect the level of mRNA encoding a tumor protein, which is alsoindicative of the presence or absence of a cancer. In general, ahematological malignancy-related tumor sequence should be present at alevel that is at least three fold higher in tumor tissue than in normaltissue

There are a variety of assay formats known to those of ordinary skill inthe art for using a binding agent to detect polypeptide markers in asample. See, e.g., Harlow and Lane, Antibodies: A Laboratory Manual,Cold Spring Harbor Laboratory, 1988. In general, the presence or absenceof a cancer in a patient may be determined by (a) contacting abiological sample obtained from a patient with a binding agent; (b)detecting in the sample a level of polypeptide that binds to the bindingagent; and (c) comparing the level of polypeptide with a predeterminedcut-off value.

In a preferred embodiment, the assay involves the use of binding agentimmobilized on a solid support to bind to and remove the polypeptidefrom the remainder of the sample. The bound polypeptide may then bedetected using a detection reagent that contains a reporter group andspecifically binds to the binding agent/polypeptide complex. Suchdetection reagents may comprise, for example, a binding agent thatspecifically binds to the polypeptide or an antibody or other agent thatspecifically binds to the binding agent, such as an anti-immunoglobulin,protein G, protein A or a lectin. Alternatively, a competitive assay maybe utilized, in which a polypeptide is labeled with a reporter group andallowed to bind to the immobilized binding agent after incubation of thebinding agent with the sample. The extent to which components of thesample inhibit the binding of the labeled polypeptide to the bindingagent is indicative of the reactivity of the sample with the immobilizedbinding agent. Suitable polypeptides for use within such assays includefull length hematological malignancy-related tumor proteins and portionsthereof to which the binding agent binds, as described above.

The solid support may be any material known to those of ordinary skillin the art to which the tumor protein may be attached. For example, thesolid support may be a test well in a microtiter plate or anitrocellulose or other suitable membrane. Alternatively, the supportmay be a bead or disc, such as glass, fiberglass, latex or a plasticmaterial such as polystyrene or polyvinylchloride. The support may alsobe a magnetic particle or a fiber optic sensor, such as those disclosed,for example, in U.S. Pat. No. 5,359,681. The binding agent may beimmobilized on the solid support using a variety of techniques known tothose of skill in the art, which are amply described in the patent andscientific literature. In the context of the present invention, the term“immobilization” refers to both noncovalent association, such asadsorption, and covalent attachment (which may be a direct linkagebetween the agent and functional groups on the support or may be alinkage by way of a cross-liking agent). Immobilization by adsorption toa well in a microtiter plate or to a membrane is preferred. In suchcases, adsorption may be achieved by contacting the binding agent, in asuitable buffer, with the solid support for a suitable amount of time.The contact time varies with temperature, but is typically between about1 hour and about 1 day. In general, contacting a well of a plasticmicrotiter plate (such as polystyrene or polyvinylchloride) with anamount of binding agent ranging from about 10 ng to about 10 μg, andpreferably about 100 ng to about 1 μg, is sufficient to immobilize anadequate amount of binding agent.

Covalent attachment of binding agent to a solid support may generally beachieved by first reacting the support with a bifunctional reagent thatwill react with both the support and a functional group, such as ahydroxyl or amino group, on the binding agent. For example, the bindingagent may be covalently attached to supports having an appropriatepolymer coating using benzoquinone or by condensation of an aldehydegroup on the support with an amine and an active hydrogen on the bindingpartner (see, e.g., Pierce Immunotechnology Catalog and Handbook, 1991,at A12-A13).

In certain embodiments, the assay is a two-antibody sandwich assay. Thisassay may be performed by first contacting an antibody that has beenimmobilized on a solid support, commonly the well of a microtiter plate,with the sample, such that polypeptides within the sample are allowed tobind to the immobilized antibody. Unbound sample is then removed fromthe immobilized polypeptide-antibody complexes and a detection reagent(preferably a second antibody capable of binding to a different site onthe polypeptide) containing a reporter group is added. The amount ofdetection reagent that remains bound to the solid support is thendetermined using a method appropriate for the specific reporter group.

More specifically, once the antibody is immobilized on the support asdescribed above, the remaining protein binding sites on the support aretypically blocked. Any suitable blocking agent known to those ofordinary skill in the art, such as bovine serum albumin or Tween 20™(Sigma Chemical Co., St. Louis, Mo.). The immobilized antibody is thenincubated with the sample, and polypeptide is allowed to bind to theantibody. The sample may be diluted with a suitable diluent, such asphosphate-buffered saline (PBS) prior to incubation. In general, anappropriate contact time (i.e., incubation time) is a period of timethat is sufficient to detect the presence of polypeptide within a sampleobtained from an individual with hematological malignancy. Preferably,the contact time is sufficient to achieve a level of binding that is atleast about 95% of that achieved at equilibrium between bound andunbound polypeptide. Those of ordinary skill in the art will recognizethat the time necessary to achieve equilibrium may be readily determinedby assaying the level of binding that occurs over a period of time. Atroom temperature, an incubation time of about 30 minutes is generallysufficient.

Unbound sample may then be removed by washing the solid support with anappropriate buffer, such as PBS containing 0.1% Tween 20™. The secondantibody, which contains a reporter group, may then be added to thesolid support. Preferred reporter groups include those groups recitedabove.

The detection reagent is then incubated with the immobilizedantibody-polypeptide complex for an amount of time sufficient to detectthe bound polypeptide. An appropriate amount of time may generally bedetermined by assaying the level of binding that occurs over a period oftime. Unbound detection reagent is then removed and bound detectionreagent is detected using the reporter group. The method employed fordetecting the reporter group depends upon the nature of the reportergroup. For radioactive groups, scintillation counting orautoradiographic methods are generally appropriate. Spectroscopicmethods may be used to detect dyes, luminescent groups and fluorescentgroups. Biotin may be detected using avidin, coupled to a differentreporter group (commonly a radioactive or fluorescent group or anenzyme). Enzyme reporter groups may generally be detected by theaddition of substrate (generally for a specific period of time),followed by spectroscopic or other analysis of the reaction products.

To determine the presence or absence of a cancer, such as hematologicalmalignancy, the signal detected from the reporter group that remainsbound to the solid support is generally compared to a signal thatcorresponds to a predetermined cut-off value. In one preferredembodiment, the cut-off value for the detection of a cancer is theaverage mean signal obtained when the immobilized antibody is incubatedwith samples from patients without the cancer. In general, a samplegenerating a signal that is three standard deviations above thepredetermined cut-off value is considered positive for the cancer. In analternate preferred embodiment, the cut-off value is determined using aReceiver Operator Curve, according to the method of Sackett et al.,Clinical Epidemiology: A Basic Science for Clinical Medicine, LittleBrown and Co., 1985, p. 106-7. Briefly, in this embodiment, the cut-offvalue may be determined from a plot of pairs of true positive rates(i.e., sensitivity) and false positive rates (100%-specificity) thatcorrespond to each possible cut-off value for the diagnostic testresult. The cut-off value on the plot that is the closest to the upperleft-hand corner (i.e., the value that encloses the largest area) is themost accurate cut-off value, and a sample generating a signal that ishigher than the cut-off value determined by this method may beconsidered positive. Alternatively, the cut-off value may be shifted tothe left along the plot, to minimize the false positive rate, or to theright, to minimize the false negative rate. In general, a samplegenerating a signal that is higher than the cut-off value determined bythis method is considered positive for a cancer.

In a related embodiment, the assay is performed in a flow-through orstrip test format, wherein the binding agent is immobilized on amembrane, such as nitrocellulose. In the flow-through test, polypeptideswithin the sample bind to the immobilized binding agent as the samplepasses through the membrane. A second, labeled binding agent then bindsto the binding agent-polypeptide complex as a solution containing thesecond binding agent flows through the membrane. The detection of boundsecond binding agent may then be performed as described above. In thestrip test format, one end of the membrane to which binding agent isbound is immersed in a solution containing the sample. The samplemigrates along the membrane through a region containing second bindingagent and to the area of immobilized binding agent. Concentration ofsecond binding agent at the area of immobilized antibody indicates thepresence of a cancer. Typically, the concentration of second bindingagent at that site generates a pattern, such as a line, that can be readvisually. The absence of such a pattern indicates a negative result. Ingeneral, the amount of binding agent immobilized on the membrane isselected to generate a visually discernible pattern when the biologicalsample contains a level of polypeptide that would be sufficient togenerate a positive signal in the two-antibody sandwich assay, in theformat discussed above. Preferred binding agents for use in such assaysare antibodies and antigen-binding fragments thereof. Preferably, theamount of antibody immobilized on the membrane ranges from about 25 ngto about 1 μg, and more preferably from about 50 ng to about 500 ng.Such tests can typically be performed with a very small amount ofbiological sample.

Of course, numerous other assay protocols exist that are suitable foruse with the tumor proteins or binding agents of the present invention.The above descriptions are intended to be exemplary only. For example,it will be apparent to those of ordinary skill in the art that the aboveprotocols may be readily modified to use hematologicalmalignancy-related tumor polypeptides to detect antibodies that bind tosuch polypeptides in a biological sample. The detection of suchhematological malignancy-related tumor protein specific antibodies maycorrelate with the presence of a cancer.

A cancer may also, or alternatively, be detected based on the presenceof T cells that specifically react with a hematologicalmalignancy-related tumor protein in a biological sample. Within certainmethods, a biological sample comprising CD4⁺ and/or CD8⁺ T cellsisolated from a patient is incubated with a hematologicalmalignancy-related tumor polypeptide, a polynucleotide encoding such apolypeptide and/or an APC that expresses at least an immunogenic portionof such a polypeptide, and the presence or absence of specificactivation of the T cells is detected. Suitable biological samplesinclude, but are not limited to, isolated T cells. For example, T cellsmay be isolated from a patient by routine techniques (such as byFicoll/Hypaque density gradient centrifugation of peripheral bloodlymphocytes). T cells may be incubated in vitro for 2-9 days (typically4 days) at 37° C. with polypeptide (e.g., 5-25 μg/ml). It may bedesirable to incubate another aliquot of a T cell sample in the absenceof hematological malignancy-related tumor polypeptide to serve as acontrol. For CD4⁺ T cells, activation is preferably detected byevaluating proliferation of the T cells. For CD8⁺ T cells, activation ispreferably detected by evaluating cytolytic activity. A level ofproliferation that is at least two fold greater and/or a level ofcytolytic activity that is at least 20% greater than in disease-freepatients indicates the presence of a cancer in the patient.

As noted above, a cancer may also, or alternatively, be detected basedon the level of mRNA encoding a hematological malignancy-related tumorprotein in a biological sample. For example, at least twooligonucleotide primers may be employed in a polymerase chain reaction(PCR) based assay to amplify a portion of a hematologicalmalignancy-related tumor cDNA derived from a biological sample, whereinat least one of the oligonucleotide primers is specific for (i.e.,hybridizes to) a polynucleotide encoding the hematologicalmalignancy-related tumor protein. The amplified cDNA is then separatedand detected using techniques well known in the art, such as gelelectrophoresis. Similarly, oligonucleotide probes that specificallyhybridize to a polynucleotide encoding a hematologicalmalignancy-related tumor protein may be used in a hybridization assay todetect the presence of polynucleotide encoding the tumor protein in abiological sample.

To permit hybridization under assay conditions, oligonucleotide primersand probes should comprise an oligonucleotide sequence that has at leastabout 60%, preferably at least about 75% and more preferably at leastabout 90%, identity to a portion of a polynucleotide encoding ahematological malignancy-related tumor protein that is at least 10nucleotides, and preferably at least 20 nucleotides, in length.Preferably, oligonucleotide primers and/or probes hybridize to apolynucleotide encoding a polypeptide described herein under moderatelystringent conditions, as defined above. Oligonucleotide primers and/orprobes which may be usefully employed in the diagnostic methodsdescribed herein preferably are at least 10-40 nucleotides in length. Ina preferred embodiment, the oligonucleotide primers comprise at least 10contiguous nucleotides, more preferably at least 15 contiguousnucleotides, of a DNA molecule having a sequence disclosed in thisapplication. Techniques for both PCR based assays and hybridizationassays are well known in the art (see, for example, Mullis et al., ColdSpring Harbor Symp. Quant. Biol., 51:263, 1987; Erlich ed., PCRTechnology, Stockton Press, NY, 1989).

One preferred assay employs RT-PCR, in which PCR is applied inconjunction with reverse transcription. Typically, RNA is extracted froma biological sample, such as biopsy tissue, and is reverse transcribedto produce cDNA molecules. PCR amplification using at least one specificprimer generates a cDNA molecule, which may be separated and visualizedusing, for example, gel electrophoresis. Amplification may be performedon biological samples taken from a test patient and from an individualwho is not afflicted with a cancer. The amplification reaction may beperformed on several dilutions of cDNA spanning two orders of magnitude.A two-fold or greater increase in expression in several dilutions of thetest patient sample as compared to the same dilutions of thenon-cancerous sample is typically considered positive.

In another embodiment, the compositions described herein may be used asmarkers for the progression of cancer. In this embodiment, assays asdescribed above for the diagnosis of a cancer may be performed overtime, and the change in the level of reactive polypeptide(s) orpolynucleotide(s) evaluated. For example, the assays may be performedevery 24-72 hours for a period of 6 months to 1 year, and thereafterperformed as needed. In general, a cancer is progressing in thosepatients in whom the level of polypeptide or polynucleotide detectedincreases over time. In contrast, the cancer is not progressing when thelevel of reactive polypeptide or polynucleotide either remains constantor decreases with time.

Certain in vivo diagnostic assays may be performed directly on a tumor.One such assay involves contacting tumor cells with a binding agent. Thebound binding agent may then be detected directly or indirectly via areporter group. Such binding agents may also be used in histologicalapplications. Alternatively, polynucleotide probes may be used withinsuch applications.

As noted above, to improve sensitivity, multiple hematologicalmalignancy-related tumor protein markers may be assayed within a givensample. It will be apparent that binding agents specific for differentproteins provided herein may be combined within a single assay. Further,multiple primers or probes may be used concurrently. The selection oftumor protein markers may be based on routine experiments to determinecombinations that results in optimal sensitivity. In addition, oralternatively, assays for tumor proteins provided herein may be combinedwith assays for other known tumor antigens.

4.24 Preparation of DNA Sequences

Certain nucleic acid sequences of cDNA molecules encoding portions ofhematological malignancy-related antigens were isolated by PCR™-basedsubtraction. This technique serves to normalize differentially expressedcDNAs, facilitating the recovery of rare transcripts, and also has theadvantage of permitting enrichment of cDNAs with small amounts of polyARNA material and without multiple rounds of hybridization. To obtainantigens overexpressed in non-Hodgkin's lymphomas, two subtractions wereperformed with a tester library prepared from a pool of three T cellnon-Hodgkin's lymphoma mRNAs. The two libraries were independentlysubtracted with different pools of driver cDNAs. Driver #1 containedcDNA prepared from specific normal tissues (lymph node, bone marrow, Tcells, heart and brain), and this subtraction generated the libraryTCS-D1 (cell non-Hodgkin's lymphoma subtracted library with driver #1).Driver #2 contained non-specific normal tissues (colon, large intestine,lung, pancreas, spinal cord, skeletal muscle, liver, kidney, skin andbrain), and this subtraction generated the library TCS-D2 (T cellnon-Hodgkin's lymphoma subtraction library with driver #2). Two othersubtractions were performed with a tester library prepared from a poolof three B cell non-Hodgkin's lymphoma mRNAs. The two libraries wereindependently subtracted with different pools of driver cDNAs. Driver #1contained cDNA prepared from specific normal tissues (lymph node, bonemarrow, B cells, heart and brain), and this subtraction generated thelibrary BCNHL/D1 (B cell non-Hodgkin's lymphoma subtracted library withdriver #1). Driver #2 contained non-specific normal tissues (brain,lung, pancreas, spinal cord, skeletal muscle, colon, spleen, largeintestine and PBMC), and this subtraction generated the library BCNHL/D2(B cell non-Hodgkin's lymphoma subtraction library with driver #2).PCR™-amplified pools were generated from the subtracted libraries andclones were sequenced. Hematological malignancy-related antigensequences may be further characterized using any of a variety of wellknown techniques. For example, PCR™ amplified clones may be arrayed ontoglass slides for microarray analysis. To determine tissue distribution,the arrayed clones may be used as targets to be hybridized withdifferent first strand cDNA probes, including lymphoma probes, leukemiaprobes and probes from different normal tissues. Leukemia and lymphomaprobes may be generated from cryopreserved samples obtained at the timeof diagnosis from NHL, Hodgkin's disease, AML, CML, CLL, ALL, MDS andmyeloma patients with poor outcome (patients who failed to achievecomplete remission following conventional chemotherapy or relapsed) orgood outcome (patients who achieved long term remission). To analyzegene expression during hematopoetic differentiation, probes may begenerated from >95% pure fractions of CD34+, CD2+, CD14+, CD15+ andCD19+ cells derived from healthy individuals.

Polynucleotide variants may generally be prepared by any method known inthe art, including chemical synthesis by, for example, solid phasephosphoramidite chemical synthesis. Modifications in a polynucleotidesequence may also be introduced using standard mutagenesis techniques,such as oligonucleotide-directed site-specific mutagenesis (see Adelmanet al., DNA 2:183, 1983). Alternatively, RNA molecules may be generatedby in vitro or in vivo transcription of DNA sequences, provided that theDNA is incorporated into a vector with a suitable RNA polymerasepromoter (such as T7 or SP6). Certain portions may be used to prepare anencoded polypeptide, as described herein. In addition, or alternatively,a portion may be administered to a patient such that the encodedpolypeptide is generated in vivo (e.g. by transfectingantigen-presenting cells, such as dendritic cells, with a cDNA constructencoding a hematological malignancy-related antigen, and administeringthe transfected cells to the patient).

A portion of a sequence complementary to a coding sequence (i.e., anantisense polynucleotide) may also be used as a probe or to modulatehematological malignancy-related antigen expression. cDNA constructsthat can be transcribed into antisense RNA may also be introduced intocells or tissues to facilitate the production of antisense RNA. Anantisense polynucleotide may be used, as described herein, to inhibitexpression of a hematological malignancy-related antigen. Antisensetechnology can be used to control gene expression through triple-helixformation, which compromises the ability of the double helix to opensufficiently for the binding of polymerases, transcription factors orregulatory molecules (see Gee et al., In Huber and Carr, Molecular andImmunologic Approaches, Futura Publishing Co. (Mt. Kisco, N.Y.; 1994)).Alternatively, an antisense molecule may be designed to hybridize with acontrol region of a gene (e.g., promoter, enhancer or transcriptioninitiation site), and block transcription of the gene; or to blocktranslation by inhibiting binding of a transcript to ribosomes.

A portion of a coding sequence or of a complementary sequence may alsobe designed as a probe or primer to detect gene expression. Probes maybe labeled with a variety of reporter groups, such as radionuclides andenzymes, and are preferably at least 10 nucleotides in length, morepreferably at least 20 nucleotides in length and still more preferablyat least 30 nucleotides in length. Primers, as noted above, arepreferably 22-30 nucleotides in length.

Any polynucleotide may be further modified to increase stability invivo. Possible modifications include, but are not limited to, theaddition of flanking sequences at the 5′ and/or 3′ ends; the use ofphosphorothioate or 2′ O-methyl rather than phosphodiesterase linkagesin the backbone; and/or the inclusion of nontraditional bases such asinosine, queosine and wybutosine, as well as acetyl- methyl-, thio- andother modified forms of adenine, cytidine, guanine, thymine and uridine.

Hematological malignancy-related antigen polynucleotides may be joinedto a variety of other nucleotide sequences using established recombinantDNA techniques. For example, a polynucleotide may be cloned into any ofa variety of cloning vectors, including plasmids, phagemids, lambdaphage derivatives and cosmids. Vectors of particular interest includeexpression vectors, replication vectors, probe generation vectors andsequencing vectors. In general, a vector will contain an origin ofreplication functional in at least one organism, convenient restrictionendonuclease sites and one or more selectable markers. Other elementswill depend upon the desired use, and will be apparent to those ofordinary skill in the art.

Within certain embodiments, polynucleotides may be formulated so as topermit entry into a cell of a mammal, and expression therein. Suchformulations are particularly useful for therapeutic purposes, asdescribed below. Those of ordinary skill in the art will appreciate thatthere are many ways to achieve expression of a polynucleotide in atarget cell, and any suitable method may be employee For example, apolynucleotide may be incorporated into a viral vector such as, but notlimited to, adenovirus, adeno-associated virus, retrovirus, or vacciniaor other pox virus (e.g., avian pox virus). Techniques for incorporatingDNA into such vectors are well known to those of ordinary skill in theart. A retroviral vector may additionally transfer or incorporate a genefor a selectable marker (to aid in the identification or selection oftransduced cells) and/or a targeting moiety, such as a gene that encodesa ligand for a receptor on a specific target cell, to render the vectortarget specific. Targeting may also be accomplished using an antibody,by methods known to those of ordinary skill in the art.

Other formulations for therapeutic purposes include colloidal dispersionsystems, such as macromolecule complexes, nanocapsules, microspheres,beads, and lipid-based systems including oil-in-water emulsions,micelles, mixed micelles, and liposomes. A preferred colloidal systemfor use as a delivery vehicle in vitro and in vivo is a liposome (i.e.,an artificial membrane vesicle). The preparation and use of such systemsis well known in the art.

4.25 Therapeutic Methods

In further aspects of the present invention, the compositions describedherein may be used for immunotherapy of hematological malignanciesincluding adult and pediatric AML, CML, ALL, CLL, myelodysplasticsyndromes (MDS), myeloproliferative syndromes (MPS), secondary leukemia,multiple myeloma, Hodgkin's lymphoma and Non-Hodgkin's lymphomas. Inaddition, compositions described herein may be used for therapy ofdiseases associated with an autoimmune response against hematopoeticprecursor cells, such as severe aplastic anemia.

Immunotherapy may be performed using any of a variety of techniques, inwhich compounds or cells provided herein function to removehematological malignancy-related antigen-expressing cells from apatient. Such removal may take place as a result of enhancing orinducing an immune response in a patient specific for hematologicalmalignancy-related antigen or a cell expressing hematologicalmalignancy-related antigen. Alternatively, hematologicalmalignancy-related antigen-expressing cells may be removed ex vivo(e.g., by treatment of autologous bone marrow, peripheral blood or afraction of bone marrow or peripheral blood). Fractions of bone marrowor peripheral blood may be obtained using any standard technique in theart.

Within such methods, pharmaceutical compositions and vaccines aretypically administered to a patient. As used herein, a “patient” refersto any warm-blooded animal, preferably a human. A patient may or may notbe afflicted with a hematological malignancy. Accordingly, the abovepharmaceutical compositions and vaccines may be used to prevent thedevelopment of a malignancy or to treat a patient afflicted with amalignancy. A hematological malignancy may be diagnosed using criteriagenerally accepted in the art. Pharmaceutical compositions and vaccinesmay be administered either prior to or following surgical removal ofprimary tumors and/or treatment such as administration of radiotherapyor conventional chemotherapeutic drugs, or bone marrow transplantation(autologous, allogeneic or syngeneic).

Within certain embodiments, immunotherapy may be active immunotherapy,in which treatment relies on the in vivo stimulation of the endogenoushost immune system to react against tumors with the administration ofimmune response-modifying agents (such as polypeptides andpolynucleotides as provided herein).

Within other embodiments, immunotherapy may be passive immunotherapy, inwhich treatment involves the delivery of agents with establishedtumor-immune reactivity (such as effector cells or antibodies) that candirectly or indirectly mediate antitumor effects and does notnecessarily depend on an intact host immune system. Examples of effectorcells include T cells as discussed above, T lymphocytes (such as CD8⁺cytotoxic T lymphocytes and CD4⁺ T-helper tumor-infiltratinglymphocytes), killer cells (such as Natural Killer cells andlymphokine-activated killer cells), B cells and antigen-presenting cells(such as dendritic cells and macrophages) expressing a polypeptideprovided herein. T cell receptors and antibody receptors specific forthe polypeptides recited herein may be cloned, expressed and transferredinto other vectors or effector cells for adoptive immunotherapy. Thepolypeptides provided herein may also be used to generate antibodies oranti-idiotypic antibodies (as described above and in U.S. Pat. No.4,918,164) for passive immunotherapy.

Effector cells may generally be obtained in sufficient quantities foradoptive immunotherapy by growth in vitro, as described herein. Cultureconditions for expanding single antigen-specific effector cells toseveral billion in number with retention of antigen recognition in vivoare well known in the art. Such in vitro culture conditions typicallyuse intermittent stimulation with antigen, often in the presence ofcytokines (such as IL-2) and non-dividing feeder cells. As noted above,immunoreactive polypeptides as provided herein may be used to rapidlyexpand antigen-specific T cell cultures in order to generate asufficient number of cells for immunotherapy. In particular,antigen-presenting cells, such as dendritic, macrophage or B cells, maybe pulsed with immunoreactive polypeptides or transfected with one ormore polynucleotides using standard techniques well known in the art.For example, antigen-presenting cells can be transfected with apolynucleotide having a promoter appropriate for increasing expressionin a recombinant virus or other expression system. Cultured effectorcells for use in therapy must be able to grow and distribute widely, andto survive long term in vivo. Studies have shown that cultured effectorcells can be induced to grow in vivo and to survive long term insubstantial numbers by repeated stimulation with antigen supplementedwith IL-2 (see, for example, Cheever et al., Immunological Reviews157:177, 1997).

Alternatively, a vector expressing a polypeptide recited herein may beintroduced into antigen presenting cells taken from a patient andclonally propagated ex vivo for transplant back into the same patient.Transfected cells may be reintroduced into the patient using any meansknown in the art, preferably in sterile form by intravenous,intracavitary, intraperitoneal or intratumor administration.

The compositions provided herein may be used alone or in combinationwith conventional therapeutic regimens such as surgery, irradiation,chemotherapy and/or bone marrow transplantation (autologous, syngeneic,allogeneic or unrelated). As discussed in greater detail below, bindingagents and T cells as provided herein may be used for purging ofautologous stem cells. Such purging may be beneficial prior to, forexample, bone marrow transplantation or transfusion of blood orcomponents thereof. Binding agents, T cells, antigen presenting cells(APC) and compositions provided herein may further be used for expandingand stimulating (or priming) autologous, allogeneic, syngeneic orunrelated hematological malignancy-related antigen-specific T-cells invitro and/or in vivo. Such hematological malignancy-relatedantigen-specific T cells may be used, for example, within donorlymphocyte infusions.

Routes and frequency of administration of the therapeutic compositionsdescribed herein, as well as dosage, will vary from individual toindividual, and may be readily established using standard techniques. Ingeneral, the pharmaceutical compositions and vaccines may beadministered by injection (e.g., intracutaneous, intramuscular,intravenous or subcutaneous), intranasally (e.g., by aspiration) ororally. Preferably, between 1 and 10 doses may be administered over a 52week period. Preferably, 6 doses are administered, at intervals of 1month, and booster vaccinations may be given periodically thereafter.Alternate protocols may be appropriate for individual patients. Asuitable dose is an amount of a compound that, when administered asdescribed above, is capable of promoting an anti-tumor immune response,and is at least 10-50% above the basal (i.e., untreated) level. Suchresponse can be monitored by measuring the anti-tumor antibodies in apatient or by vaccine-dependent generation of cytolytic effector cellscapable of killing the patient's tumor cells in vitro. Such vaccinesshould also be capable of causing an immune response that leads to animproved clinical outcome (e.g. more frequent remissions, complete orpartial or longer disease-free survival) in vaccinated patients ascompared to non-vaccinated patients. In general, for pharmaceuticalcompositions and vaccines comprising one or more polypeptides, theamount of each polypeptide present in a dose ranges from about 100 μg to5 mg per kg of host. Suitable dose sizes will vary with the size of thepatient, but will typically range from about 0.1 mL to about 5 mL.

In general, an appropriate dosage and treatment regimen provides theactive compound(s) in an amount sufficient to provide therapeutic and/orprophylactic benefit. Such a response can be monitored by establishingan improved clinical outcome (e.g., more frequent remissions, completeor partial, or longer disease-free survival) in treated patients ascompared to non-treated patients. Increases in preexisting immuneresponses to a hematological malignancy-related antigen generallycorrelate with an improved clinical outcome. Such immune responses maygenerally be evaluated using standard proliferation, cytotoxicity orcytokine assays, which may be performed using samples obtained from apatient before and after treatment.

Within further aspects, methods for inhibiting the development of amalignant disease associated with hematological malignancy-relatedantigen expression involve the administration of autologous T cells thathave been activated in response to a hematological malignancy-relatedantigen polypeptide or hematological malignancy-relatedantigen-expressing APC, as described above. Such T cells may be CD4⁺and/or CD8⁺, and may be proliferated as described above. The T cells maybe administered to the individual in an amount effective to inhibit thedevelopment of a malignant disease. Typically, about 1×10⁹ to 1×10¹¹ Tcells/M² are administered intravenously, intracavitary or in the bed ofa resected tumor. It will be evident to those skilled in the art thatthe number of cells and the frequency of administration will bedependent upon the response of the patient.

Within certain embodiments, T cells may be stimulated prior to anautologous bone marrow transplantation. Such stimulation may take placein vivo or in vitro. For in vitro stimulation, bone marrow and/orperipheral blood (or a fraction of bone marrow or peripheral blood)obtained from a patient may be contacted with a hematologicalmalignancy-related antigen polypeptide, a polynucleotide encoding ahematological malignancy-related antigen polypeptide and/or an APC thatexpresses a hematological malignancy-related antigen polypeptide underconditions and for a time sufficient to permit the stimulation of Tcells as described above. Bone marrow, peripheral blood stem cellsand/or hematological malignancy-related antigen-specific T cells maythen be administered to a patient using standard techniques.

Within related embodiments, T cells of a related or unrelated donor maybe stimulated prior to a syngeneic or allogeneic (related or unrelated)bone marrow transplantation. Such stimulation may take place in vivo orin vitro. For in vitro stimulation, bone marrow and/or peripheral blood(or a fraction of bone marrow or peripheral blood) obtained from arelated or unrelated donor may be contacted with a hematologicalmalignancy-related antigen polypeptide, hematological malignancy-relatedantigen polynucleotide and/or APC that expresses a hematologicalmalignancy-related antigen polypeptide under conditions and for a timesufficient to permit the stimulation of T cells as described above. Bonemarrow, peripheral blood stem cells and/or hematologicalmalignancy-related antigen-specific T cells may then be administered toa patient using standard techniques.

Within other embodiments, hematological malignancy-relatedantigen-specific T cells, antibodies or antigen-binding fragmentsthereof as described herein may be used to remove cells expressinghematological malignancy-related antigen from a biological sample, suchas autologous bone marrow, peripheral blood or a fraction of bone marrowor peripheral blood (e.g., CD34⁺ enriched peripheral blood (PB) prior toadministration to a patient). Such methods may be performed bycontacting the biological sample with such T cells, antibodies orantibody fragments under conditions and for a time sufficient to permitthe reduction of hematological malignancy-related antigen expressingcells to less than 10%, preferably less than 5% and more preferably lessthan 1%, of the total number of myeloid or lymphatic cells in the bonemarrow or peripheral blood. Such contact may be achieved, for example,using a column to which antibodies are attached using standardtechniques. Antigen-expressing cells are retained on the column. Theextent to which such cells have been removed may be readily determinedby standard methods such as, for example, qualitative and quantitativePCR analysis, morphology, immunohistochemistry and FACS analysis. Bonemarrow or PB (or a fraction thereof) may then be administered to apatient using standard techniques.

4.26 Diagnostic Methods

In general, a hematological malignancy may be detected in a patientbased on the presence of hematological malignancy-related antigen and/orpolynucleotide in a biological sample (such as blood, sera, urine and/ortumor biopsies) obtained from the patient. In other words, hematologicalmalignancy-related antigens may be used as a marker to indicate thepresence or absence of such a malignancy. The binding agents providedherein generally permit detection of the level of antigen that binds tothe agent in the biological sample. Polynucleotide primers and probesmay be used to detect the level of mRNA encoding hematologicalmalignancy-related antigen, which is also indicative of the presence orabsence of a hematological malignancy. In general, hematologicalmalignancy-related antigen should be present at a level that is at leastthree fold higher in a sample obtained from a patient afflicted with ahematological malignancy than in the sample obtained from an individualnot so afflicted.

There are a variety of assay formats known to those of ordinary skill inthe art for using a binding agent to detect polypeptide markers in asample. See, e.g., Harlow and Lane, Antibodies: A Laboratory Manual,Cold Spring Harbor Laboratory, 1988. In general, the presence or absenceof a hematological malignancy in a patient may be determined by (a)contacting a biological sample obtained from a patient with a bindingagent; (b) detecting in the sample a level of polypeptide that binds tothe binding agent; and (c) comparing the level of polypeptide with apredetermined cut-off value.

In a preferred embodiment, the assay involves the use of binding agentimmobilized on a solid support to bind to and remove the polypeptidefrom the remainder of the sample. The bound polypeptide may then bedetected using a detection reagent that contains a reporter group andspecifically binds to the binding agent/polypeptide complex. Suchdetection reagents may comprise, for example, a binding agent thatspecifically binds to the polypeptide or an antibody or other agent thatspecifically binds to the binding agent, such as an anti-immunoglobulin,protein G, protein A or a lectin. Alternatively, a competitive assay maybe utilized, in which a polypeptide is labeled with a reporter group andallowed to bind to the immobilized binding agent after incubation of thebinding agent with the sample. The extent to which components of thesample inhibit the binding of the labeled polypeptide to the bindingagent is indicative of the reactivity of the sample with the immobilizedbinding agent. Suitable polypeptides for use within such assays includefull length hematological malignancy-related antigens and portionsthereof to which the binding agent binds, as described above.

The solid support may be any material known to those of ordinary skillin the art to which the hematological malignancy-related antigenpolypeptide may be attached. For example, the solid support may be atest well in a microtiter plate or a nitrocellulose or other suitablemembrane. Alternatively, the support may be a bead or disc, such asglass, fiberglass, latex or a plastic material such as polystyrene orpolyvinylchloride. The support may also be a magnetic particle or afiber optic sensor, such as those disclosed, for example, in U.S. Pat.No. 5,359,681. The binding agent may be immobilized on the solid supportusing a variety of techniques known to those of skill in the art, whichare amply described in the patent and scientific literature. In thecontext of the present invention, the term “immobilization” refers toboth noncovalent association, such as adsorption, and covalentattachment (which may be a direct linkage between the agent andfunctional groups on the support or may be a linkage by way of across-linking agent). Immobilization by adsorption to a well in amicrotiter plate or to a membrane is preferred. In such cases,adsorption may be achieved by contacting the binding agent, in asuitable buffer, with the solid support for a suitable amount of time.The contact time varies with temperature, but is typically between about1 hour and about 1 day. In general, contacting a well of a plasticmicrotiter plate (such as polystyrene or polyvinylchloride) with anamount of binding agent ranging from about 10 ng to about 10 μg, andpreferably about 100 ng to about 1 μg, is sufficient to immobilize anadequate amount of binding agent.

Covalent attachment of binding agent to a solid support may generally beachieved by first reacting the support with a bifunctional reagent thatwill react with both the support and a functional group, such as ahydroxyl or amino group, on the binding agent. For example, the bindingagent may be covalently attached to supports having an appropriatepolymer coating using benzoquinone or by condensation of an aldehydegroup on the support with an amine and an active hydrogen on the bindingpartner (see, e.g., Pierce Immunotechnology Catalog and Handbook, 1991,at A12-A13).

In certain embodiments, the assay is a two-antibody sandwich assay. Thisassay may be performed by first contacting an antibody that has beenimmobilized on a solid support, commonly the well of a microtiter plate,with the sample, such that polypeptides within the sample are allowed tobind to the immobilized antibody. Unbound sample is then removed fromthe immobilized polypeptide-antibody complexes and a detection reagent(preferably a second antibody capable of binding to a different site onthe polypeptide) containing a reporter group is added. The amount ofdetection reagent that remains bound to the solid support is thendetermined using a method appropriate for the specific reporter group.

More specifically, once the antibody is immobilized on the support asdescribed above, the remaining protein binding sites on the support aretypically blocked. Any suitable blocking agent known to those ofordinary skill in the art, such as bovine serum albumin or Tween 20™(Sigma Chemical Co., St. Louis, Mo.). The immobilized antibody is thenincubated with the sample, and polypeptide is allowed to bind to theantibody. The sample may be diluted with a suitable diluent, such asphosphate-buffered saline (PBS) prior to incubation. In general, anappropriate contact time (i.e., incubation time) is a period of timethat is sufficient to detect the presence of polypeptide within a sampleobtained from an individual with a hematological malignancy. Preferably,the contact time is sufficient to achieve a level of binding that is atleast about 95% of that achieved at equilibrium between bound andunbound polypeptide. Those of ordinary skill in the art will recognizethat the time necessary to achieve equilibrium may be readily determinedby assaying the level of binding that occurs over a period of time. Atroom temperature, an incubation time of about 30 minutes is generallysufficient.

Unbound sample may then be removed by washing the solid support with anappropriate buffer, such as PBS containing 0.1% Tween 20™. The secondantibody, which contains a reporter group, may then be added to thesolid support. Preferred reporter groups include those groups recitedabove.

The detection reagent is then incubated with the immobilizedantibody-polypeptide complex for an amount of time sufficient to detectthe bound polypeptide. An appropriate amount of time may generally bedetermined by assaying the level of binding that occurs over a period oftime. Unbound detection reagent is then removed and bound detectionreagent is detected using the reporter group. The method employed fordetecting the reporter group depends upon the nature of the reportergroup. For radioactive groups, scintillation counting orautoradiographic methods are generally appropriate. Spectroscopicmethods may be used to detect dyes, luminescent groups and fluorescentgroups. Biotin may be detected using avidin, coupled to a differentreporter group (commonly a radioactive or fluorescent group or anenzyme). Enzyme reporter groups may generally be detected by theaddition of substrate (generally for a specific period of time),followed by spectroscopic or other analysis of the reaction products.

To determine the presence or absence of a hematological malignancy, thesignal detected from the reporter group that remains bound to the solidsupport is generally compared to a signal that corresponds to apredetermined cut-off value. In one preferred embodiment, the cut-offvalue for the detection of a hematological malignancy is the averagemean signal obtained when the immobilized antibody is incubated withsamples from patients without the malignancy. In general, a samplegenerating a signal that is three standard deviations above thepredetermined cut-off value is considered positive for the malignancy.In an alternate preferred embodiment, the cut-off value is determinedusing a Receiver Operator Curve, according to the method of Sackett etal., Clinical Epidemiology: A Basic Science for Clinical Medicine,Little Brown and Co., 1985, p. 106-7. Briefly, in this embodiment, thecut-off value may be determined from a plot of pairs of true positiverates (i.e., sensitivity) and false positive rates (100%-specificity)that correspond to each possible cut-off value for the diagnostic testresult. The cut-off value on the plot that is the closest to the upperleft-hand corner (i.e., the value that encloses the largest area) is themost accurate cut-off value, and a sample generating a signal that ishigher than the cut-off value determined by this method may beconsidered positive. Alternatively, the cut-off value may be shifted tothe left along the plot, to minimize the false positive rate, or to theright, to minimize the false negative rate. In general, a samplegenerating a signal that is higher than the cut-off value determined bythis method is considered positive for a malignancy.

In a related embodiment, the assay is performed in a flow-through orstrip test format, wherein the binding agent is immobilized on amembrane, such as nitrocellulose. In the flow-through test, polypeptideswithin the sample bind to the immobilized binding agent as the samplepasses through the membrane. A second, labeled binding agent then bindsto the binding agent-polypeptide complex as a solution containing thesecond binding agent flows through the membrane. The detection of boundsecond binding agent may then be performed as described above. In thestrip test format, one end of the membrane to which binding agent isbound is immersed in a solution containing the sample. The samplemigrates along the membrane through a region containing second bindingagent and to the area of immobilized binding agent. Concentration ofsecond binding agent at the area of immobilized antibody indicates thepresence of a hematological malignancy. Typically, the concentration ofsecond binding agent at that site generates a pattern, such as a line,that can be read visually. The absence of such a pattern indicates anegative result. In general, the amount of binding agent immobilized onthe membrane is selected to generate a visually discernible pattern whenthe biological sample contains a level of polypeptide that would besufficient to generate a positive signal in the two-antibody sandwichassay, in the format discussed above. Preferred binding agents for usein such assays are antibodies and antigen-binding fragments thereof.Preferably, the amount of antibody immobilized on the membrane rangesfrom about 25 ng to about 1 μg, and more preferably from about 50 ng toabout 500 ng. Such tests can typically be performed with a very smallamount of biological sample.

Of course, numerous other assay protocols exist that are suitable foruse with the hematological malignancy-related antigen sequences orbinding agents of the present invention. The above descriptions areintended to be exemplary only. For example, it will be apparent to thoseof ordinary skill in the art that the above protocols may be readilymodified to use hematological malignancy-related antigen polypeptides todetect antibodies that bind to such polypeptides in a biological sample.The detection of hematological malignancy-related antigen-specificantibodies may correlate with the presence of a hematological.

A malignancy may also, or alternatively, be detected based on thepresence of T cells that specifically react with hematologicalmalignancy-related antigen in a biological sample. Within certainmethods, a biological sample comprising CD4⁺ and/or CD8⁺ T cellsisolated from a patient is incubated with a hematologicalmalignancy-related antigen polypeptide, a polynucleotide encoding such apolypeptide and/or an APC that expresses such a polypeptide, and thepresence or absence of specific activation of the T cells is detected.Suitable biological samples include, but are not limited to, isolated Tcells. For example, T cells may be isolated from a patient by routinetechniques (such as by Ficoll/Hypaque density gradient centrifugation ofperipheral blood lymphocytes). T cells may be incubated in vitro for 2-9days (typically 4 days) at 37° C. with Mtb-81 or Mtb-67.2 polypeptide(e.g., 5-25 μg/ml). It may be desirable to incubate another aliquot of aT cell sample in the absence of hematological malignancy-related antigenpolypeptide to serve as a control. For CD4⁺ T cells, activation ispreferably detected by evaluating proliferation of the T cells. For CD8⁺T cells, activation is preferably detected by evaluating cytolyticactivity. A level of proliferation that is at least two fold greaterand/or a level of cytolytic activity that is at least 20% greater thanin disease-free patients indicates the presence of a hematologicalmalignancy in the patient.

As noted above, a hematological malignancy may also, or alternatively,be detected based on the level of mRNA encoding hematologicalmalignancy-related antigen in a biological sample. For example, at leasttwo oligonucleotide primers may be employed in a polymerase chainreaction (PCR) based assay to amplify a portion of hematologicalmalignancy-related antigen cDNA derived from a biological sample,wherein at least one of the oligonucleotide primers is specific for(i.e., hybridizes to) a polynucleotide encoding the hematologicalmalignancy-related antigen protein. The amplified cDNA is then separatedand detected using techniques well known in the art, such as gelelectrophoresis. Similarly, oligonucleotide probes that specificallyhybridize to a polynucleotide encoding hematological malignancy-relatedantigen may be used in a hybridization assay to detect the presence ofpolynucleotide encoding hematological malignancy-related antigen in abiological sample.

To permit hybridization under assay conditions, oligonucleotide primersand probes should comprise an oligonucleotide sequence that has at leastabout 60%, preferably at least about 75% and more preferably at leastabout 90%, identity to a portion of a polynucleotide encodinghematological malignancy-related antigen that is at least 10nucleotides, and preferably at least 20 nucleotides, in length.Preferably, oligonucleotide primers and/or probes hybridize to apolynucleotide encoding a polypeptide described herein under moderatelystringent conditions, as defined above. Oligonucleotide primers and/orprobes which may be usefully employed in the diagnostic methodsdescribed herein preferably are at least 10-40 nucleotides in length.Techniques for both PCR based assays and hybridization assays are wellknown in the art (see, for example, Mullis et al., Cold Spring HarborSymp. Quant. Biol., 51:263, 1987; Erlich ed., PCR Technology, StocktonPress, NY, 1989).

One preferred assay employs RT-PCR, in which PCR is applied inconjunction with reverse transcription. Typically, RNA is extracted froma biological sample such as a biopsy tissue and is reverse transcribedto produce cDNA molecules. PCR amplification using at least one specificprimer generates a cDNA molecule, which may be separated and visualizedusing, for example, gel electrophoresis. Amplification may be performedon biological samples taken from a test patient and from an individualwho is not afflicted with a hematological malignancy. The amplificationreaction may be performed on several dilutions of cDNA spanning twoorders of magnitude. A two-fold or greater increase in expression inseveral dilutions of the test patient sample as compared to the samedilutions of the sample from a normal individual is typically consideredpositive.

In preferred embodiments, such assays may be performed using samplesenriched for cells expressing the hematological malignancy-relatedantigen(s) of interest. Such enrichment may be achieved, for example,using a binding agent as provided herein to remove the cells from theremainder of the biological sample. The removed cells may then beassayed as described above for biological samples.

In further embodiments, hematological malignancy-related antigens may beused as markers for monitoring disease progression or the response totherapy of a hematological malignancy. In this embodiment, assays asdescribed above for the diagnosis of a hematological malignancy may beperformed over time, and the change in the level of reactivepolypeptide(s) evaluated. For example, the assays may be performed every24-72 hours for a period of 6 months to 1 year, and thereafter performedas needed. In general, a malignancy is progressing in those patients inwhom the level of polypeptide detected by the binding agent increasesover time. In contrast, the malignancy is not progressing when the levelof reactive polypeptide either remains constant or decreases with time.

Certain in vivo diagnostic assays may be performed directly on a tumor.One such assay involves contacting tumor cells with a binding agent. Thebound binding agent may then be detected directly or indirectly via areporter group. Such binding agents may also be used in histologicalapplications. Alternatively, polynucleotide probes may be used withinsuch applications.

As noted above, to improve sensitivity, multiple markers may be assayedwithin a given sample. It will be apparent that binding agents specificfor different proteins provided herein may be combined within a singleassay. Further, multiple primers or probes may be used concurrently. Theselection of markers may be based on routine experiments to determinecombinations that results in optimal sensitivity.

Further diagnostic applications include the detection of extramedullarydisease (e.g., cerebral infiltration of blasts in leukemias). Withinsuch methods, a binding agent may be coupled to a tracer substance, andthe diagnosis is performed in vivo using well known techniques. Coupledbinding agent may be administered as described above, and extramedullarydisease may be detected based on assaying the presence of tracersubstance. Alternatively, a tracer substance may be associated with a Tcell specific for hematological malignancy-related antigen, permittingdetection of extramedullary disease based on assays to detect thelocation of the tracer substance.

4.27 Exemplary Definitions

In accordance with the present invention, nucleic acid sequencesinclude, but are not limited to, DNAs (including and not limited togenomic or extragenomic DNAs), genes, peptide nucleic acids (PNAs) RNAs(including, but not limited to, rRNAs, mRNAs and tRNAs), nucleosides,and suitable nucleic acid segments either obtained from native sources,chemically synthesized, modified, or otherwise prepared in whole or inpart by the hand of man.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although any methods andcompositions similar or equivalent to those described herein can be usedin the practice or testing of the present invention, the preferredmethods and compositions are described herein. For purposes of thepresent invention, the following terms are defined below:

A, an: In accordance with long standing patent law convention, the words“a” and “an” when used in this application, including the claims,denotes “one or more”.

Expression: The combination of intracellular processes, includingtranscription and translation undergone by a polynucleotide such as astructural gene to synthesize the encoded peptide or polypeptide.

Promoter: a term used to generally describe the region or regions of anucleic acid sequence that regulates transcription.

Regulatory Element: a term used to generally describe the region orregions of a nucleic acid sequence that regulates transcription.

Structural gene: A gene or sequence region that is expressed to producean encoded peptide or polypeptide.

Transformation: A process of introducing an exogenous polynucleotidesequence (e.g., a vector, a recombinant DNA or RNA molecule) into a hostcell or protoplast in which that exogenous nucleic acid segment isincorporated into at least a first chromosome or is capable ofautonomous replication within the transformed host cell. Transfection,electroporation, and naked nucleic acid uptake all represent examples oftechniques used to transform a host cell with one or morepolynucleotides.

Transformed cell: A host cell whose nucleic acid complement has beenaltered by the introduction of one or more exogenous polynucleotidesinto that cell.

Transgenic cell: Any cell derived or regenerated from a transformed cellor derived from a transgenic cell, or from the progeny or offspring ofany generation of such a transformed host cell.

Transgenic animal: An animal or a progeny or an offspring of anygeneration thereof that is derived from a transformed animal cell,wherein the animal's DNA contains an introduced exogenous nucleic acidmolecule not originally present in a native, wild type, non-transgenicanimal of the same species. The terms “transgenic animal” and“transformed animal” have sometimes been used in the art as synonymousterms to define an animal, the genetic contents of which has beenmodified to contain one or more exogenous nucleic acid segments.

Vector: A nucleic acid molecule, typically comprised of DNA, capable ofreplication in a host cell and/or to which another nucleic acid segmentcan be operatively linked so as to bring about replication of theattached segment. A plasmid, cosmid, or a virus is an exemplary vector.

The terms “substantially corresponds to”, “substantially homologous”, or“substantial identity” as used herein denotes a characteristic of anucleic acid or an amino acid sequence, wherein a selected nucleic acidor amino acid sequence has at least about 70 or about 75 percentsequence identity as compared to a selected reference nucleic acid oramino acid sequence. More typically, the selected sequence and thereference sequence will have at least about 76, 77, 78, 79, 80, 81, 82,83, 84 or even 85 percent sequence identity, and more preferably atleast about 86, 87, 88, 89, 90, 91, 92, 93, 94, or 95 percent sequenceidentity. More preferably still, highly homologous sequences often sharegreater than at least about 96, 97, 98, or 99 percent sequence identitybetween the selected sequence and the reference sequence to which it wascompared. The percentage of sequence identity may be calculated over theentire length of the sequences to be compared, or may be calculated byexcluding small deletions or additions which total less than about 25percent or so of the chosen reference sequence. The reference sequencemay be a subset of a larger sequence, such as a portion of a gene orflanking sequence, or a repetitive portion of a chromosome. However, inthe case of sequence homology of two or more polynucleotide sequences,the reference sequence will typically comprise at least about 18-25nucleotides, more typically at least about 26 to 35 nucleotides, andeven more typically at least about 40, 50, 60, 70, 80, 90, or even 100or so nucleotides. Desirably, which highly homologous fragments aredesired, the extent of percent identity between the two sequences willbe at least about 80%, preferably at least about 85%, and morepreferably about 90% or 95% or higher, as readily determined by one ormore of the sequence comparison algorithms well-known to those of skillin the art, such as e.g., the FASTA program analysis described byPearson and Lipman (1988).

The term “naturally occurring” as used herein as applied to an objectrefers to the fact that an object can be found in nature. For example, apolypeptide or polynucleotide sequence that is present in an organism(including viruses) that can be isolated from a source in nature andwhich has not been intentionally modified by the hand of man in alaboratory is naturally-occurring. As used herein, laboratory strains ofrodents that may have been selectively bred according to classicalgenetics are considered naturally occurring animals.

As used herein, a “heterologous” is defined in relation to apredetermined referenced gene sequence. For example, with respect to astructural gene sequence, a heterologous promoter is defined as apromoter which does not naturally occur adjacent to the referencedstructural gene, but which is positioned by laboratory manipulation.Likewise, a heterologous gene or nucleic acid segment is defined as agene or segment that does not naturally occur adjacent to the referencedpromoter and/or enhancer elements.

“Transcriptional regulatory element” refers to a polynucleotide sequencethat activates transcription alone or in combination with one or moreother nucleic acid sequences. A transcriptional regulatory element can,for example, comprise one or more promoters, one or more responseelements, one or more negative regulatory elements, and/or one or moreenhancers.

As used herein, a “transcription factor recognition site” and a“transcription factor binding site” refer to a polynucleotidesequence(s) or sequence motif(s) which are identified as being sites forthe sequence-specific interaction of one or more transcription factors,frequently taking the form of direct protein-DNA binding. Typically,transcription factor binding sites can be identified by DNAfootprinting, gel mobility shift assays, and the like, and/or can bepredicted on the basis of known consensus sequence motifs, or by othermethods known to those of skill in the art.

As used herein, the term “operably linked” refers to a linkage of two ormore polynucleotides or two or more nucleic acid sequences in afunctional relationship. A nucleic acid is “operably linked” when it isplaced into a functional relationship with another nucleic acidsequence. For instance, a promoter or enhancer is operably linked to acoding sequence if it affects the transcription of the coding sequence.Operably linked means that the DNA sequences being linked are typicallycontiguous and, where necessary to join two protein coding regions,contiguous and in reading frame. However, since enhancers generallyfunction when separated from the promoter by several kilobases andintronic sequences may be of variable lengths, some polynucleotideelements may be operably linked but not contiguous.

“Transcriptional unit” refers to a polynucleotide sequence thatcomprises at least a first structural gene operably linked to at least afirst cis-acting promoter sequence and optionally linked operably to oneor more other cis-acting nucleic acid sequences necessary for efficienttranscription of the structural gene sequences, and at least a firstdistal regulatory element as may be required for the appropriatetissue-specific and developmental transcription of the structural genesequence operably positioned under the control of the promoter and/orenhancer elements, as well as any additional cis sequences that arenecessary for efficient transcription and translation (e.g.,polyadenylation site(s), mRNA stability controlling sequence(s), etc.

As noted above, the present invention is generally directed tocompositions and methods for using the compositions, for example in thetherapy and diagnosis of cancer, such as hematological malignancy.Certain illustrative compositions described herein include hematologicalmalignancy-related tumor polypeptides, polynucleotides encoding suchpolypeptides, binding agents such as antibodies, antigen presentingcells (APCs) and/or immune system cells (e.g., T cells). A“hematological malignancy-related tumor protein,” as the term is usedherein, refers generally to a protein that is expressed in hematologicmalignancy-related tumor cells at a level that is at least two fold, andpreferably at least five fold, greater than the level of expression in anormal tissue, as determined using a representative assay providedherein. Certain hematological malignancy-related tumor proteins aretumor proteins that react detectably (within an immunoassay, such as anELISA or Western blot) with antisera of a patient afflicted withhematological malignancy.

4.28 Biological Functional Equivalents

Modification and changes may be made in the structure of thepolynucleotides and peptides of the present invention and still obtain afunctional molecule that encodes a peptide with desirablecharacteristics, or still obtain a genetic construct with the desirableexpression specificity and/or properties. As it is often desirable tointroduce one or more mutations into a specific polynucleotide sequence,various means of introducing mutations into a polynucleotide or peptidesequence known to those of skill in the art may be employed for thepreparation of heterologous sequences that may be introduced into theselected cell or animal species. In certain circumstances, the resultingencoded peptide sequence is altered by this mutation, or in other cases,the sequence of the peptide is unchanged by one or more mutations in theencoding polynucleotide. In other circumstances, one or more changes areintroduced into the promoter and/or enhancer regions of thepolynucleotide constructs to alter the activity, or specificity of theexpression elements and thus alter the expression of the heterologoustherapeutic nucleic acid segment operably positioned under the controlof the elements.

When it is desirable to alter the amino acid sequence of one or more ofthe heterologous peptides encoded by the expression construct to createan equivalent, or even an improved, second-generation molecules, theamino acid changes may be achieved by changing one or more of the codonsof the encoding DNA sequence, according to Table 1.

For example, certain amino acids may be substituted for other aminoacids in a protein structure without appreciable loss of interactivebinding capacity with structures such as, for example, antigen-bindingregions of antibodies or binding sites on substrate molecules. Since itis the interactive capacity and nature of a protein that defines thatprotein's biological functional activity, certain amino acid sequencesubstitutions can be made in a protein sequence, and, of course, itsunderlying DNA coding sequence, and nevertheless obtain a protein withlike properties. It is thus contemplated by the inventors that variouschanges may be made in the peptide sequences of the disclosedcompositions, or corresponding DNA sequences which encode said peptideswithout appreciable loss of their biological utility or activity. TABLE1 Amino Acids Codons Alanine Ala A GCA GCC GCG GCU Cysteine Cys C UGCUGU Aspartic acid Asp D GAC GAU Glutamic acid Glu E GAA GAGPhenylalanine Phe F UUC UUU Glycine Gly G GGA GGC GGG GGU Histidine HisH CAC CAU Isoleucine Ile I AUA AUC AUU Lysine Lys K AAA AAG Leucine LeuL UUA UUG CUA CUC CUG CUU Methionine Met M AUG Asparagine Asn N AAC AAUProline Pro P CCA CCC CCG CCU Glutamine Gln Q CAA CAG Arginine Arg R AGAAGG CGA CGC CGG CGU Serine Ser S AGC AGU UCA UCC UCG UCU Threonine Thr TACA ACC ACG ACU Valine Val V GUA GUC GUG GUU Tryptophan Trp W UGGTyrosine Tyr Y UAC UAU

In making such changes, the hydropathic index of amino acids may beconsidered. The importance of the hydropathic amino acid index inconferring interactive biologic function on a protein is generallyunderstood in the art (Kyte and Doolittle, 1982, incorporate herein byreference). It is accepted that the relative hydropathic character ofthe amino acid contributes to the secondary structure of the resultantprotein, which in turn defines the interaction of the protein with othermolecules, for example, enzymes, substrates, receptors, DNA, antibodies,antigens, and the like. Each amino acid has been assigned a hydropathicindex on the basis of their hydrophobicity and charge characteristics(Kyte and Doolittle, 1982), these are: isoleucine (+4.5); valine (+4.2);leucine (+3.8); phenylalanine (+2.8); cysteine/cystine (+2.5);methionine (+1.9); alanine (+1.8); glycine (−0.4); threonine (−0.7);serine (−0.8); tryptophan (−0.9); tyrosine (−1.3); proline (−1.6);histidine (−3.2); glutamate (−3.5); glutamine (−3.5); aspartate (−3.5);asparagine (−3.5); lysine (−3.9); and arginine (4.5).

It is known in the art that certain amino acids may be substituted byother amino acids having a similar hydropathic index or score and stillresult in a protein with similar biological activity, i.e. still obtaina biological functionally equivalent protein. In making such changes,the substitution of amino acids whose hydropathic indices are within ±2is preferred, those that are within ±1 are particularly preferred, andthose within ±0.5 are even more particularly preferred. It is alsounderstood in the art that the substitution of like amino acids can bemade effectively on the basis of hydrophilicity. U.S. Pat. No.4,554,101, incorporated herein by reference, states that the greatestlocal average hydrophilicity of a protein, as governed by thehydrophilicity of its adjacent amino acids, correlates with a biologicalproperty of the protein.

As detailed in U.S. Pat. No. 4,554,101, the following hydrophilicityvalues have been assigned to amino acid residues: arginine (+3.0);lysine (+3.0); aspartate (+3.0±1); glutamate (+3.0±1); serine (+0.3);asparagine (+0.2); glutamine (+0.2); glycine (0); threonine (−0.4);proline (−0.5±1); alanine (−0.5); histidine (−0.5); cysteine (−1.0);methionine (−1.3); valine (−1.5); leucine (−1.8); isoleucine (−1.8);tyrosine (−2.3); phenylalanine (−2.5); tryptophan (−3.4). It isunderstood that an amino acid can be substituted for another having asimilar hydrophilicity value and still obtain a biologically equivalent,and in particular, an immunologically equivalent protein. In suchchanges, the substitution of amino acids whose hydrophilicity values arewithin ±2 is preferred, those that are within ±1 are particularlypreferred, and those within ±0.5 are even more particularly preferred.

As outlined above, amino acid substitutions are generally thereforebased on the relative similarity of the amino acid side-chainsubstituents, for example, their hydrophobicity, hydrophilicity, charge,size, and the like. Exemplary substitutions which take several of theforegoing characteristics into consideration are well known to those ofskill in the art and include: arginine and lysine; glutamate andaspartate; serine and threonine; glutamine and asparagine; and valine,leucine and isoleucine.

5. EXAMPLES

The following examples are included to demonstrate preferred embodimentsof the invention. However, those of skill in the art should, in light ofthe present disclosure, appreciate that many changes can be made in thespecific embodiments which are disclosed and still obtain a like orsimilar result without departing from the spirit and scope of theinvention described in the appended claims.

5.1 Example 1 Identification of Hematological Malignancy-Related AntigenPolynucleotides

This Example illustrates the identification of hematologicalmalignancy-related antigen polynucleotides from non-Hodgkin's lymphomas.

Hematological malignancy-related antigen polynucleotides were isolatedby PCR-based subtraction. PolyA mRNA was prepared from T cellnon-Hodgkin's lymphomas, B cell non-Hodgkin's lymphomas and normaltissues. Six cDNA libraries were constructed, PCR-subtracted andanalyzed. Two libraries were constructed using pools of three T cellnon-Hodgkin's lymphoma mRNAs (referred to herein as TCS libraries). Twoothers were constructed using pools of three B cell non-Hodgkin'slymphoma mRNAs (referred to herein as BCNHL libraries). Two otherlibraries were constructed using a pool of 2 Hodgkin's lymphoma mRNAs(referred to herein as HLS libraries. cDNA synthesis, hybridization andPCR amplification were performed according to Clontech's user manual(PCR-Select cDNA Subtraction), with the following changes: 1) cDNA wasrestricted with a mixture of enzymes, including MscI, PvuII, StuI andDraI, instead of the single enzyme RsaI; and 2) the ratio of driver totester cDNA was increased in the hybridization steps (to 76:1) to give amore stringent subtraction.

The two TCS libraries were independently subtracted with different poolsof driver cDNAs. Driver #1 contained cDNA prepared from specific normaltissues (lymph node, bone marrow, T cells, heart and brain), and thissubtraction generated the library TCS-D1 (T cell non-Hodgkin's lymphomasubtracted library with driver #1). Driver #2 contained non-specificnormal tissues (colon, large intestine, lung, pancreas, spinal cord,skeletal muscle, liver, kidney, skin and brain), and this subtractiongenerated the library TCS-D2 (T cell non-Hodgkin's lymphoma subtractionlibrary with driver #2).

Similarly, the two BCNHL libraries were independently subtracted withdifferent pools of driver cDNAs. Driver #1 contained cDNA prepared fromspecific normal tissues (lymph node, bone marrow, B cells, heart andbrain), and this subtraction generated the library BCNHL/D1 (B cellnon-Hodgkin's lymphoma subtracted library with driver #1). Driver #2contained non-specific normal tissues (brain, lung, pancreas, spinalcord, skeletal muscle, colon, spleen, large intestine and PBMC), andthis subtraction generated the library BCNHL/D2 (B cell non-Hodgkin'slymphoma subtraction library with driver #2).

The two HLS libraries were independently subtracted with different poolsof driver cDNAs. Driver #1 contained cDNA prepared from specific normaltissues (lymph node, bone marrow, B cells and lung) and this subtractiongenerated HLS-D1 (Hodgkin's lymphoma subtraction library with driver#1). Driver #2 contained non-specific normal tissues (colon, largeintestine, lung, pancreas, spinal cord, skeletal muscle, liver, kidney,skin and brain) and this generated the library HLS-D2 (Hodgkin'slymphoma subtraction library with driver #2).

To analyze the efficiency of the subtraction, actin (a housekeepinggene) was PCR amplified from dilutions of subtracted as well asunsubtracted PCR samples. Furthermore, the complexity and redundancy ofeach library was characterized by sequencing 96 clones from each of thePCR subtraction libraries (TCS-D1, TCS-D2, BCNHL/D1, BCNHL/D2, HLS-D1and HLS-D2). These analyses indicated that the libraries are enrichedfor genes overexpressed in leukemia tissues and specifically T cell andB cell non-Hodgkin's lymphoma and M. Hodgkin's lymphoma samples.

Following PCR amplification, the cDNAs were cloned into the pCR2.1-TOPOplasmid vector (Invitrogen).

Sequences obtained from these analyses were searched against knownsequences in the publicly available databases using the BLAST 2.0release. The default BLAST parameters used were as follows: GAPPARAMETERS: Open Gap=0, Extended Gap=0; OUTPUT PARAMETERS: Expect=10.0,Threshold=0, Number of Alignments=250; For BLASTN, the search parameterswere as follows: Mismatch=−3, Reward=1, Word size=0. The alignments werepresented pair-wise, with a window percent identity=22. All availableprotein and nucleotide databases were searched, including, PIR,SwissPROT, GenBank, Mouse EST, Human EST, Other EST, Human repeat andhigh throughput sequences, and published patents and patent applicationdatabase.

From these, a number of unique sequences were identified thatrepresented novel polynucleotide sequences that had not previously beendescribed in the GenBank and other sequence databases. A number of othersequences were identified that appeared to contain significant homologywith one or more sequences previously identified in the databases,although they were described only as genomic or cDNA clones, and had noknown function. The remaining sequences corresponded to known genes. Theclones obtained from this analysis are summarized in Tables 2-6 inco-pending application U.S. Ser. No. 09/796,692.

5.2 Example 2 Analysis of Subtracted cDNA Sequences by MicroarrayAnalysis

Subtracted cDNA sequences were analyzed by microarray analysis toevaluate their expression in hematological malignancies and normaltissues. Using this approach, cDNA sequences were PCR amplified andtheir mRNA expression profiles in hematological malignancies and normaltissues are examined using cDNA microarray technology essentially asdescribed (Shena et al., 1995).

In brief, the clones identified from the subtracted cDNA librariesanalyses were immobilized and arrayed onto glass slides as multiplereplicas on microarray slides and the slides were hybridized with twodifferent sets of probes, with each location on the microarray slidecorresponding to a unique cDNA clone (as many as 5500 clones can bearrayed on a single slide, or chip). Each chip is hybridized with a pairof cDNA probes that are fluorescence-labeled with Cy3 and Cy5,respectively. The set of probes derived from the hematologicalmalignancies was labeled with cy3 while the other set of probes derivedfrom a pool of normal tissues was labeled with cy5. Typically, 1 μg ofpolyA⁺ RNA was used to generate each cDNA probe. After hybridization,the chips were scanned and the fluorescence intensity recorded for bothCy3 and Cy5 channels. The difference in intensities (i.e., cy3/cy5ratios) following hybridization with both probe sets provided theinformation on the relative expression level of each cDNA sequencesimmobilized on the slide in tumor versus normal tissues. There aremultiple built-in quality control steps. First, the probe quality ismonitored using a panel of ubiquitously expressed genes. Secondly, thecontrol plate also can include yeast DNA fragments of whichcomplementary RNA may be spiked into the probe synthesis for measuringthe quality of the probe and the sensitivity of the analysis. Thismethodology provides a sensitivity of 1 in 100,000 copies of mRNA, andthe reproducibility of the technology may be ensured by includingduplicated control cDNA elements at different locations.

Analysis of hematological malignancy subtracted clones by microarrayanalyses on a variety of microarray chips identified the sequences setforth in SEQ ID NO:1 through SEQ ID NO:668 of co-pending applicationU.S. Ser. No. 09/796,692 as being at least two-fold overexpressed inhematological malignancies versus normal tissues.

5.3 Example 3 Polynucleotide and Polypeptide Compositions: BriefDescription of the cDNA Clones and Open Reading Frames Identified bySubtractive Hybridization and Microarray Analysis

Table 7 in co-pending application U.S. Ser. No. 09/796,692 lists thesequences of the polynucleotides obtained during the analyses of thepresent invention. Shown are the 668 polynucleotide sequences, alongwith their clone name identifiers, as well as the serial number andfiling date of the priority provisional patent application in which theclone was first identified.

Table 8 in co-pending application U.S. Ser. No. 09/796,692 identifiesthe putative open reading frames obtained from analyses of the cDNAsequences obtained in SEQ ID NO:1-SEQ ID NO:668 in the co-pendingapplication. Shown are the sequence identifiers, the clone name andtranslation frame, and the start and stop nucleotides in thecorresponding DNA sequence used to generate the polypeptide sequence ofthe open reading frame.

Table 9 in co-pending application U.S. Ser. No. 09/796,692 identifies anadditional set of particular hematological malignancy-related cDNAsequences that were obtained using the subtractive library andmicroarray methods as described above. These sequences, designated SEQID NO:2533-SEQ ID NO:9597 in the co-pending application, are shown inthe Table along with the original clone name, and the serial number andfiling date of the priority provisional application in which the clonewas first described.

5.4 Example 4 Identification of a Specific Gene, Ly1448P, Associatedwith B Cell Leukemias, Lymphomas and Multiple Myelomas

SEQ ID NO:9599 in co-pending application U.S. Ser. No. 10/040,862, alsotermed “Ly1448P,” a portion of which was disclosed earlier in co-pendingapplication U.S. Ser. No. 09/96,692 as SEQ ID NO:636 was identified in aseries of MicroArray analyses as described in Examples 1-3 in sections5.1-5.3 of co-pending application U.S. Ser. No. 09/796,692.Oligonucleotides directed against a portion of SEQ ID NO:9599 were usedin a series of RealTime PCR experiments using RNAs isolated from normalcells and hematologically malignant cells. SEQ ID NO:9599 appeared to beexpressed in normal B cell lines, CD 19⁺ cell lines, and highlyexpressed in a subset of Non-Hodgkin's B-cell lymphoma cell lines,Hodgkin's lymphoma cell lines, follicular lymphoma cell lines, andChronic Lymphocytic Leukemia cell lines.

SEQ ID NO:9599, which is a 523 base pair cDNA fragment, was used toscreen the LIFESEQ® Gold database and two additional clones wereidentified, SEQ ID NO:9598, also termed “LS 1384258.1”, which is a 622base pair cDNA fragment, and SEQ ID NO:9600, also termed “LS 368109.1”,which is a 1,908 base pair cDNA.

The BLAST analysis determined that the open reading frame (ORF) of SEQID NO: 9600 begins at nucleotide 777, ends at nucleotide 1562 andencodes a 261 amino acid protein as identified in SEQ ID NO:9611, termedLy1448P protein. Further analysis of SEQ ID NO:9600 using both TMpredand PSORTII indicated that SEQ ID NO:9611 is a type-1b membrane protein,containing a predicted transmembrane domain beginning at amino acid 156and ending at amino acid 177. The extracellular portion of SEQ IDNO:9611 has homology with immunoglobulins and contains a predictedIg-like domain. The intracellular portion of SEQ ID NO:9611 contains SrcHomology-2 (SH2) binding domains an Immune Receptor Tyrosine-BasedInhibition Motif (ITIM) and an Immune Receptor Tyrosine-based ActivationMotif (ITAM). As such, Ly1448P protein may play a specific role inhematopoetic cell signaling. Further structural and homology analysis ofthese cDNA fragments is described in the co-pending application U.S.Ser. No. 10/040,862 and are discussed below.

The Blast analysis also showed that Ly1448P shares homology with twoother protein families, the SPAP1 family and the IRTA family. Ly1448Pshares homology with the SPAP1 family of proteins which consists of 3members, SPAP1a, SPAP1b, and SPAP1c. As discussed in more detail below,the SPAP1 proteins identified all have a unique 42 amino acid terminus.SPAP1b has a unique 18 amino acid carboxy terminus and SPAP1c has aunique 9 amino acid carboxy terminus. Ly1448P also shares homology withthe IRTA family of proteins and specifically IRTA 4. As discussed inmore detail below, Ly1448P and IRTA4 share multiple exons and theproteins identified appear to arise from alternative splicing events.

5.5 Example 5 Identification of Ly1448 Immungenic Peptides

This example illustrates the identification of peptides useful forgenerating cytotoxic T cell or helper T cell responses.

Ly1448P protein peptides useful for generating a cellular immuneresponse were identified. Preferred epitope binding motifs for many ofthe HLA class I and class II molecules are known. The Ly1448P amino acidsequence was searched for peptides that would bind to the specific HLAmolecules identified in Table 2. 9mer peptides predicted to bind to eachof the specific HLA molecules were identified and SEQ ID NOscorresponding to the peptides which bind to each HLA molecule areindicated. TABLE 2 Ly1448P CTL Peptides Identified HLA molecule selectedLy1448P peptides identified HLA_0201 SEQ ID NOs: 9618-9637 HLA A_0205SEQ ID NOs: 9638-9657 HLA A24 SEQ ID NOs: 9658-9697 HLA A3 SEQ ID NOs:9698-9717 HLA A68.1 SEQ ID NOs: 9718-9737 HLA A_1101 SEQ ID NOs:9738-9757 HLA A_3101 SEQ ID NOs: 9758-9777 HLA A_3302 SEQ ID NOs:9778-9797 HLA B14 SEQ ID NOs: 9798-9817 HLA B40 SEQ ID NOs: 9818-9837HLA B60 SEQ ID NOs: 9838-9857 HLA B61 SEQ ID NOs: 9858-9877 HLA B62 SEQID NOs: 9878-9897 HLA B7 SEQ ID NOs: 9898-9917 HLA B8 SEQ ID NOs:9918-9937 HLA B_2702 SEQ ID NOs: 9938-9957 HLA B_2705 SEQ ID NOs:9958-9977 HLA B_3501 SEQ ID NOs: 9978-9997 HLA B_3701 SEQ ID NOs:9998-10017 HLA B_3801 SEQ ID NOs: 10018-10037 HLA B_3901 SEQ ID NOs:10038-10057 HLA B_3902 SEQ ID NOs: 10038-10057 HLA B_4403 SEQ ID NOs:10058-10077 HLA B_5101 SEQ ID NOs: 10078-10097 HLA B_5102 SEQ ID NOs:10098-10117 HLA B_5103 SEQ ID NOs: 10118-10137 HLA B_5201 SEQ ID NOs:10138-10157 HLA B_5801 SEQ ID NOs: 10158-10177 HLA Cw_0301 SEQ ID NOs:10178-10197 HLA Cw_0401 SEQ ID NOs: 10198-10217 HLA Cw_0602 SEQ ID NOs:10218-10237 HLA Cw_0702 SEQ ID NOs: 10238-10257 HLA Db SEQ ID NOs:10258-10277 HLA Db_revised SEQ ID NOs: 10278-10297 HLA Dd SEQ ID NOs:10298-10317 HLA Kb SEQ ID NOs: 10318-10337 HLA Kd SEQ ID NOs:10338-10357 HLA Kk SEQ ID NOs: 10358-10377 HLA Kk SEQ ID NOs:10378-10397 HLA Ld SEQ ID NOs: 10398-10417 HLA Cattle_A20 SEQ ID NOs:10418-10437

The location of additional immunogenic peptides was determined byanalyzing the complete Ly1448P protein amino acid sequence of SEQ IDNO:9611 using the program TSITES. The results are shown in FIG. 5.TSITES identified the location of predicted amphipathic helices (A), thelocation of amino acid residues matching the Rothbard/Taylor motif (R),the location of residues matching the IAd motif (D) and the location ofresidues matching the IEd motif (d). Based on this analysis, fouradditional peptides were identified that are predicted to containepitopes that will bind to CD4⁺ T cells. Peptide 1 spans amino acids40-78 of the Ly1448P protein and is represented by SEQ ID NO:10455.Peptide 2 spans amino acids 127-150 of the Ly1448P protein and isrepresented by SEQ ID NO:10456. Peptide 3 spans amino acids 210-233 ofthe Ly1448P protein and is represented by SEQ ID NO:10457. Peptide 4spans amino acids 141-178 of the Ly1448P protein and is represented bySEQ ID NO:10458.

Ly1448P protein immunogenic peptides comprising 5 non-overlapping 31meror 32mer amino acid fragments (SEQ ID NOs:9613-9617) of theextracellular domain Ly1448P protein (amino acids 1-156 of SEQ IDNO:9611) were generated and are being used to generate antibodiesspecific for the Ly1448P protein. These antibodies will be useful inrecognizing Ly1448P protein for diagnostic and therapeutic use in bothnormal and diseased cells. Each peptide fragment used to generateantibodies also contains a GCG peptide linker sequence at thecarboxy-terminus of the fragment. These antibodies may also recognizethe IRTA4 protein. Antibodies immunoreactive to epitopes present onpeptides defined by SEQ ID NOs:9614-9617 may also recognize epitopespresent in SPAP1a.

Seventeen overlapping immunogenic peptides derived from Ly1448P proteinsequence were also generated. 16-30mer amino acid peptides weregenerated, each peptide contains sequential amino acid sequences andoverlaps peptides containing adjacent sequences by 15 amino acids (SEQID NOs:10438-10453). One peptide encoding the carboxy-terminus ofLy1448P protein is 21 amino acids long and also overlaps the adjacentpeptide by 15 amino acids (SEQ ID NO:10454). These seventeen peptideswill also be used to generate antibodies immunoreactive to epitopespresent on the peptides. Antibodies which recognize epitopes present onpeptides identified by SEQ ID NOs:10438 and 10439 will recognize Ly1448Pprotein whereas antibodies which recognize epitopes present on peptidesidentified by SEQ ID NOs:10440-10454 may recognize both Ly1448P andSPAP1a proteins. Antibodies generated against all of these peptides mayalso recognize IRTA4. Antibodies generated against these 17 overlappingpeptides will be useful for both diagnostic and therapeutic purposes.

5.6 Example 6 Immunogenic Peptides Derived from the IRTA Proteins

This example illustrates the identification of peptides useful forgenerating cytotoxic T cell or helper T cell responses.

The amino acid sequences of each of the IRTA proteins, IRTA 1, 2a, 2b,2c, 3, 4, and 5, were analysed using the program TSITES. This programidentifies epitopes suitable for binding to T Helper cells. Peptidescorresponding to each of the T Helper epitopes for each protein wereidentified. SEQ ID NOs corresponding to each of the peptides arepresented in Table 3. TABLE 3 IRTA T Helper Epitopes IRTA ProteinPeptides Identified IRTA 1 SEQ ID NOs: 11,061-11,080 IRTA 2a SEQ ID NOs:11,081-11,098 IRTA 2b SEQ ID NOs: 11,099-11,115 IRTA 2c SEQ ID NOs:11,116-11,130 IRTA 3 SEQ ID NOs: 11,131-11,147 IRTA 4 SEQ ID NOs:11,148-11,165 IRTA 5 SEQ ID NOs: 11,166-11,177

5.7 Example 7 Identification of Two Specific Genes, Ly1447 and Ly1481are Associated with B Cell Leukemias, Lymphomas and Multiple Myelomas

This example illustrates the identification of two specific genes thatare associated with B cell leukemias, lymphomas and multiple myelomas.

Ly1447 and Ly1481, portions of which were disclosed earlier inco-pending application U.S. Ser. No. 09/796,692, are cDNAs alsoassociated with B cell leukemias, lymphomas and multiple myelomas. Thenucleotide sequence of Ly1447 is disclosed herein as SEQ ID NO:9602. Thenucleotide sequence of Ly1481 is disclosed herein as SEQ ID NO:9603. Theamino acid sequence encoded by SEQ ID NO:6 is disclosed herein as SEQ IDNO:10466.

SEQ ID NOs:9602 and 9603 were used in BLASTX searches of both GenBankand GenSeq databases. Homologous sequences were only found in the GenSeqdatabase. This analysis showed that SEQ ID NO:9602 was homologous to the3′ untranslated region of the IRTA2c gene on chromosome 1. SEQ IDNO:9603 was homologous to a portion of the coding region of IRTA3 alsoon chromosome 1.

The IRTA superfamily of genes comprises IRTA 1, 2a, 2b, 2c, 3, 4, and 5,whose nucleic acid sequences are disclosed herein as SEQ IDNOs:9604-9610 respectively and whose corresponding amino acid sequencesare disclosed herein as SEQ ID NOs:10459-10465 respectively. These IRTAgenes are located on chromosome 1 (q21) and are associated withchromosomal translocations. These translocations combine a portion ofchromosome 1 with a portion of chromosome 14 (q32). This portion ofchromosome 14 is the location of the genes encoding the immunoglobulinproteins. These chromosomal translocations are associated with various Bcell leukemias, lymphomas and multiple myelomas.

5.8 Example 8 Detection of Ly1448P-Specific and TCL-1-SpecificAntibodies in Patients with Hematological Malignancies

This example illustrates that Ly1448P-specific and TCL-1-specificantibodies are present in the sera of patients with hematologicalmalignancies, such as, but not restricted to, lymphoma. Detection ofthese specific antibodies provides a tool for early diagnosis ofhematological malignancies; specifically for the screening of healthyindividuals or individuals at risk for developing lymphomas, such astransplant recipients and immunocompromised patients (i.e., AIDSpatients) for the presence of hematological malignancies or a marker formonitoring minimal residual disease. Furthermore, these data demonstratethat Ly1448P and TCL-1 are immunogenic in patients.

The specific antibodies derived from these patients can be used toidentify epitopes which can be used for the therapy of hematologicalmalignancies. FIG. 5 shows the Ly1448P specific Ab data (black andwhite).

48 control sera were screened by ELISA assay using recombinant Ly1448Pprotein or TCL-1 protein. The mean OD reading of all 48 normal sera+2.5×standard deviations was determined as a cut off level and shown in thefigures as a black line. All sera demonstrating higher values weredefined as positive.

5.9 Example 9 Ly1448P Splice Variants Associated with B Cell Leukemias,Lymphomas and Multiple Myelomas

This example illustrates the identification of all of the exons and thevarious spliced forms of the Ly1448P gene and its encoded protein thatare associated with B cell leukemias, lymphomas and multiple myelomas.

Co-pending application U.S. Ser. No. 10/040,862 describes that thenucleic acid sequence encoding Ly1448P (SEQ ID NO:9600) was used toscreen the publicly available human genome database, Genbank and privatedatabases. Additional Ly1448P sequences are presented here in SEQ IDNO:11,016. Homologous sequences were identified in the databases and onhuman chromosome 1. A BLAST search of GenBank determined that Ly1448Pprotein shared homology with three recently identified proteins, SH2domain-containing phosphatase anchor proteins 1a (SPAP1a) 1b (SPAP1b)and 1c (SPAP1c), Xu et al., Biochem. Biophys. Res. Commun. 280: 768-775(2001). These three proteins appear to be alternatively spliced forms ofLy1448P. The nucleic acid sequence encoding SPAP1a, an alternativelyspliced variant of Ly1448P, is 765 nucleotides long (SEQ ID. NO:11,036)and encodes a protein 255 amino acids long (SEQ ID NOs:9612 and 11,057).Additional SPAP1a nucleotide sequences may be found in SEQ ID NOs:9601,11,035 and Genbank Accession No. AF319438. SPAP1b is an alternativelyspliced variant of both Ly1448P and SPAP1a. The nucleic acid sequenceencoding SPAP1b is 576 bp (SEQ ID NO:11,038) and encodes a protein 192amino acids long (SEQ ID NO:11,058). Additional SPAP1b nucleotidesequences may be found in SEQ ID NO:11,037 and Genbank Accession No.AF319439. SPAP1c is an alternatively spliced variant of Ly1448P andSPAPs1a and 1b. The nucleotide sequence encoding SPAP1c is 432nucleotides long (SEQ ID NO:11292 and GenBank Acession No: AF319440) andencodes a protein 144 amino acids long (SEQ ID NO:11293).

A BLASTX search of both the GenBank and GenSeq databases indicated thatLy1448P and SPAP1a, 1b, and 1c also share homology with theImmunoglobulin Receptor Translocation Associated Protein 4 (“IRTA4”).IRTA4 also appears to be an alternatively spliced variant of Ly1448P andSPAP1a, 1b, and 1c. The nucleotide sequence encoding IRTA4 is 1524nucleotides long (SEQ ID NO:11,001), and encodes a protein 508 aminoacids long (SEQ ID NOs:10,464 and 11,039). Additional IRTA4 nucleotidesequences may be found in SEQ ID NOs:9609, 11,000 and Genbank AccessionNo. AF459633. Recently IRTA4 was also identified as a member of a familyof Fc receptor homologs, specifically FcRH2 (Davis et al., Proc. Nat.Acad. Sci. USA 98:9772-9777 (2001) and Genbank Accession No. AY043465).Family members exhibit preferential B cell expression.

PCR primers comprising nucleotide sequences 5′CTGCTGTGGTCATTGCTGGTC3′(sense) (SEQ ID NO:11,059) and 5′GACACTGGAATTCTCACAGGGATATTC3′(antisense) (SEQ ID NO:11,060) were used to amplify IRTA4 and IRTA4 likesequences from a B cell cDNA library comprising cDNAs prepared fromthree B cell non-Hodgkin's lymphoma samples. One clone, isolated andcharacterized, appeared to be another alternatively spliced version ofLy1448P and IRTA4 and is termed Patti PCR clone or FL PCR ORF Clone (SEQID NO:11,032). SEQ ID NO:11,032 is 2392 nucleotides in length andcontains two open reading frames. Open reading frame 1 (“ORF1”) (SEQ IDNO:11,033) is 420 nucleotides long and encodes a 140 amino acid protein(SEQ ID NO:11,055). Open reading frame 2 (“ORF2”) (SEQ ID NO:11,034) is783 nucleotides long and encodes a 261 amino acid protein (SEQ IDNO:11,056).

Exons and introns were identified by comparing the publicly availablechromosome 1 genomic sequence data to nucleotides encoding a Ly1448Pfragment, IRTA4, LY1448P, Patti PCR Clone, SPAP1a, 1b, and 1c, (SEQ IDNOs:9598, 11,000, 11,016, 11,032, 11,035, 11,037 and 11,292respectively) and are shown diagrammatically in FIG. 7. Open readingframes were identified using the DNASTAR™ MapDraw program (DNASTARInc.). This comparison identified 18 exons in this region ofchromosome 1. This comparison further identified alternatively usedexons and exons containing alternatively used splice donors or spliceacceptors. Sequences for each of these exons and exons containingalternatively used splice donors or acceptors are found in SEQ IDNOs:10,979-10,999 as shown in Table 4. TABLE 4 EXON NUMBER SEQ ID NO:Exon 1 SEQ ID NO: 10,979 Exon 2 SEQ ID NO: 10,980 Exon 3 SEQ ID NO:10,981 Exon 4 SEQ ID NO: 10,982 Exon 5 SEQ ID NO: 10,983 Exon 6 SEQ IDNO: 10,984 Exon 7 SEQ ID NO: 10,985 Exon 8 SEQ ID NO: 10,986 Exon 9 SEQID NO: 10,987 Exon 10 SEQ ID NO: 10,988 Exon 10GT SEQ ID NO: 10,989 ExonAG11 SEQ ID NO: 10,990 Exon 12 SEQ ID NO: 10,991 Exon AG12 SEQ ID NO:10,992 Exon 13 SEQ ID NO: 10,993 Exon 13GT SEQ ID NO: 10,994 Exon 14 SEQID NO: 10,995 Exon 15 SEQ ID NO: 10,996 Exon 16 SEQ ID NO: 10,997 Exon17 SEQ ID NO: 10,998 Exon 18 SEQ ID NO: 10,999

Exons 10 and 13 contain alternatively used splice donors and exons 11and 12 contain alternatively used splice acceptors. Full length exon 11is used in the Ly1448P fragment (SEQ ID NO:9598). All other transcriptsuse the alternative splice acceptor form of exon 11. Full length exon 12contains a translation start site and is used in all SPAP1 transcripts(splice forms r and s and SPAP1c) (SEQ ID NOs:11,035-11,038 and11,291-11,292). Splice form SPAP1c does not contain any exon 13sequences and contains 27 additional nucleotides at the 3′ end of exon12 encoding 9 additional amino acids that are found at the carboxyterminus of SPAP1c protein (SEQ ID NOs:11,293). Full length exon 13 isused in SPAP1b transcripts (splice form s) (SEQ ID NO:11,037-11,038) andcontains a transcription stop codon and polyadenylation signals. Exon 7contains a translational start site and is used in IRTA4 transcripts(splice forms a-h and q) (SEQ ID NOs:11,000-11,015 and 11,032-11,033).Exons 1-6 are used in Ly1448P transcripts (splice forms i-p) (SEQ DNOs:11,016-11,31). Exon 9 contains a translation start site for spliceforms i-p. Exon 10 using the alternative splice donor is found in thePatti PCR Clone (splice form q1) (SEQ ID NOs:11,032-11,034. Exons 13 and13GT contain the transmembrane domain. Exon 12 contains the translationstart sight for splice forms r and s. These results are also showndiagrammatically in FIGS. 8 and 9. Sequences encoding each of thepotential combinations may be found in Table 5.

The multiple alternate splice forms for genes and their encoded proteinsinclude both predicted membrane (receptor) and non-membrane molecules.For example, Patti PCR Clone ORF1 encodes a secreted non-membrane boundmolecule (SEQ ID NOs:11,032-11,033 and 11,055). Patti PCR Clone ORF1contains the extracellular domains found in exons 7-9 and 10GT and novelamino acids encoded by exon AG11 (see FIG. 9, splice form q1). SPAP1calso encodes a secreted protein (SEQ ID NOs:11,291-11,293). SPAP1ccontains the same amino terminal 135 amino acids found in SPAP1a and 1band contains a unique 9 amino acid carboxy terminus. All of thealternatively spliced molecules identified in FIG. 8 and Table 5 andshown diagrammatically in FIG. 9 have different biological activity. Byway of example, different membrane (receptor) Ly1448P molecules can havedifferent signal transduction properties dependent on the composition oftheir intracellular polypeptide sequence (the “signaling” portion of themolecule) and their extracellular polypeptide sequence (the“ligand-binding” portion of the molecule). The linking of differentextracellular domains to different intracellular sequences provides forreceptors that yield different functional signals (e.g. proliferation,differentiation, apoptosis) in response to the same or a differentligand. Non-membrane Ly1448P molecules act to modulate signal from themembrane receptor Ly1448P molecules by acting as competitors by bindingto ligand, or by binding to intracellular proteins responsible fortransducing the signal from the receptor. Such examples result in bothpositive (e.g. proliferation/differentiation) and negative (e.g.apoptosis) affects on cells expressing these proteins. Antibodiesdirected to the various forms of Ly1448P, which in turn containdifferent epitopes, modulate the biological effect of Ly1448P in asimilar manner. Probes (e.g. antibodies or oligonucleotides) directed toalternatively spliced forms of the proteins or nucleic acids are usefulas diagnostics to determine which Ly1448P variant is being expressed.These probes are useful as therapeutics to antagonize the specific formsof the Ly1448P gene or protein. TABLE 5 Nucleotide Polypeptide SpliceForm SEQ ID NO: SEQ ID NO: Ly1448P_a SEQ ID NO:11,000-11,001 SEQ IDNO:11,039 Ly1448P_b SEQ ID NO:11,002-11,003 SEQ ID NO:11,040 Ly1448P_cSEQ ID NO:11,004-11,005 SEQ ID NO:11,041 Ly1448P_d SEQ IDNO:11,006-11,007 SEQ ID NO:11,042 Ly1448P_e SEQ ID NO:11,008-11,009 SEQID NO:11,043 Ly1448P_f SEQ ID NO:11,010-11,011 SEQ ID NO:11,044Ly1448P_g SEQ ID NO:11,012-11,013 SEQ ID NO:11,045 Ly1448P_h SEQ IDNO:11,014-11,015 SEQ ID NO:11,046 Ly1448P_i SEQ ID NO:11,016-11,017 SEQID NO:11,047 Ly1448P_j SEQ ID NO:11,018-11,019 SEQ ID NO:11,048Ly1448P_k SEQ ID NO:11,020-11,021 SEQ ID NO:11,049 Ly1448P_l SEQ IDNO:11,022-11,023 SEQ ID NO:11,050 Ly1448P_m SEQ ID NO:11,024-11,025 SEQID NO:11,051 Ly1448P_n SEQ ID NO:11,026-11,027 SEQ ID NO:11,052Ly1448P_o SEQ ID NO:11,028-11,029 SEQ ID NO:11,053 Ly1448P_p SEQ IDNO:11,030-11,031 SEQ ID NO:11,054 Ly1448P_qORF1 SEQ ID NO:11,032-11,033SEQ ID NO:11,055 Ly1448P_qORF2 SEQ ID NO:11,032&11,034 SEQ ID NO:11,056Ly1448P_r SEQ ID NO:11,035-11,036 SEQ ID NO:11,057 Ly1448P_s SEQ IDNO:11,037-11,038 SEQ ID NO:11,058 SPAP1c SEQ ID NO:11,291-11,292 SEQ IDNO:11,293

5.10 Example 10 Ly1448P Specific Antibodies

This example illustrates the production of recombinant Ly1448P proteinin E. coli. This example further illustrates the production ofpolyclonal and mono-clonal anti-sera against that recombinant protein.These anti-sera are useful as diagnostics to identify tumors expressingthe Ly1448P antigen. These anti-sera are useful as therapeutics fortreating tumors expressing the Ly1448P antigen.

The nucleotide sequence encoding Ly1448P (nucleotides 780-1573 of SEQ IDNO:9600) were cloned into the E. coli expression vector pET 28 which hadbeen modified to include nucleotides encoding an initiation methionineresidue, a glutamine residue and a six amino acid histidine tag adjacentto transcription initiation sequences of the vector. PCR primerscomprising nucleotide sequences 780-801 (sense) and 1559-1573(antisense) of SEQ ID NO:9600 were used to amplify a nucleotide sequenceencoding amino acids 2-261 of SEQ ID NO:9611 using standard techniques(Sambrook et al., supra and Ausubel et al., supra). The antisense primeralso contained a XhoI restriction enzyme site to facilitate cloning. Theresulting PCR product was digested with XhoI and ligated into themodified pET 28 which had been digested with Eco 721 and XhoI. Therecombinant clone was used to transform BLR (DE3) pLys S and HMS 174pLys S bacteria. Ly1448P protein was purified from recombinant bacterialcultures. Ly1448P protein was purified from cell lysates by Ni columnchromatography (Qiagen), followed by anion exchange chromatography andanti-his affinity chromatography steps.

The resulting purified recombinant Ly1448P protein and KLH (keyholelimpet hemocyanin) conjugated, 31-mer peptides corresponding to theLY1448 amino acids 1 to 31 (SEQ ID NO:9613)(MGKKTQRSLSAELEIPAVKESDAGKYYCRAD-GCG-KLH) (peptide 1) and 63 to 93 (SEQID NO:9615) (QAAVGDLLELHCEALRGSPPILYQFYHEDVT-GCG-KLH) (peptide 3) wasinjected into rabbits using the following scheme: day 0: 0.15 mg/rabbit,in CFA, (complete Freund's adjuvant) s.c., day 21:0.15 mg/rabbit, inIFA, (incomplete Freund's adjuvant) s.c., day 35:0.15 mg/rabbit, in IFA,s.c., day 42:production bleed (˜20 mL).

For ELISA analysis of antigen specificity, rabbit sera were titeredagainst Ly1448P-coated (0.1 mg/well) 96-well microtiter plates. Sampleswere diluted initially at 1:100, and then serially diluted 3 fold andadded to the plates in duplicate. Antigen specific binding of rabbit IgGto the plate was revealed by goat anti-rabbit IgG (H+ L)-HRP conjugate,and the plates were developed with TMB substrate and read at OD450 nm ona microplate reader.

For FACS analysis, HEK293 and Ly1448P/HEK293 cells (infra) werecollected and washed with ice cold staining buffer (PBS+1% BSA+SodiumAzide). Next, the cells were resuspended in 50 ul staining buffer andincubated for 30 minutes on ice with 75 ul of rabbit anti-Ly1448Precombinant or peptide sera diluted 1:500. Pre-bleed sera was used atthe same concentration as a negative control. The cells were washed 3times with staining buffer and then incubated with a 1:100 dilution of agoat anti-rabbit Ig(H+ L)-FITC reagent (Southern Biotechnology) for 30minutes on ice. Following 3 washes, the cells were resuspended instaining buffer containing Propidium Iodide (PI), a vital stain thatallows for identification of permeable cells, and analyzed by FACS.

The results of the FACS analysis show that the polyclonal antiseraraised against the full length recombinant protein (anti-Ly1448Pantisera) and both synthetic peptides (anti-peptide anti-sera) were ableto recognize and bind to full length, native Ly1448P expressed in HEK293transfectants. Pre-immunization (pre-bleed) antisera obtained from theeach rabbit did not bind to the Ly1448P/HEK293 expressing cells.Similarly, anti-Ly1448P and anti-peptide antisera did not bind tocontrol HEK293 cells.

The results of the ELISA assys show that anti-Ly1448P antisera raisedwere able to bind strongly to immobilized recombinant Ly1448P andpeptide 1 (SEQ ID NO:9613) and bind weakly to the peptide 3 (SEQ IDNO:9615). Polyclonal antisera (anti-peptide 1) raised against thesynthetic peptide 1 (SEQ ID NO:9613) bound strongly to peptide 1 (SEQ IDNO:9613) and recombinant LY1448 protein, and did not bind at all topeptide 3 (SEQ ID NO:9615). Polyclonal antisera (anti-peptide 3) raisedagainst the synthetic peptide 3 (SEQ ID NO:9615) bound strongly topeptide 3 (SEQ ID NO:9615) and the recombinant Ly1448P protein.Anti-peptide 3 bound weakly to peptide 1. These results indicate thatthe anti-Ly1448P antisera and the anti-peptide antisera recognizeepitopes present on LY1448 and would be useful for both therapeutic anddiagnostic purposes.

Murine monoclonal antibodies specific for recombinant Ly1448P were alsogenerated. The monoclonal antibodies were raised by immunization of micewith bacterially-expressed recombinant protein Ly1448P. Conventionalantibody-secreting murine hybridomas were made by fusion of the immunespleen cells with murine myeloma cells. Ly1448P specific mAbs wereidentified in the supernatants of the hybridomas by ELISA usingrecombinant Ly1448P protein and Ly1448P-FLAG/pCEP4/HEK293 transfectants.The hybridomas were cloned and antibody secretion was stabilized.Specificity of the antibodies was confirmed by Western blot withrecombinant protein and immunohistochemistry with Ly1448P/HEKtransfectants. Epitope specificity was determined by ELISA withsynthetic peptides deduced from the sequence of Ly1448P. Thesemonoclonal antibodies are used therapeutically or as diagnostic reagentsin hematologic malignancies.

Female Balb/c mice were immunized intraperitoneally with 10-25micrograms of bacterially produced recombinant Ly1448P protein inadjuvant at intervals of 9 to 60 days. After five immunizations inadjuvant, 3 micrograms of recombinant protein were administeredintravenously in the absence of adjuvant. Two days later immune spleencells were harvested and hybridomas were made using conventionalPEG-mediated fusion and cells were distributed among several hundredmicrotiter wells and cultured under HAT selection. In approximately 2weeks a majority of wells showed growth and supernatants were harvestedand assayed by ELISA for the presence of antibody reactive torecombinant Ly1448P protein. The ELISA method was performed as follows:

Nunc MaxiSorp 96-well plates (Sigma) were coated overnight at 4° C. withpeptides at 10 μg/ml in sodium carbonate buffer (pH9.6), using 100μl/well. Ly1448P recombinant protein and an irrelevant peptide werecoated at 1 μg/ml as positive and negative controls, respectively.Plates were washed with phosphate buffered saline(PBS)/Tween (Sigma) andblocked with Super Block (250 μl/well) for 1 hour at room temperature.Plates were washed after blocking, mAbs and control muring IgG werediluted in Super Block to 1 μg/ml and added to each infividualpeptide/protein-coated well, 100 μl/well. Incubation time is 1 hour atroom temperature. Plates were washed at the end of the incubation periodwith PBS/Tween. Bound antibodies were detected by adding horse radishperoxidase (HRP) conjugated goat anti-mouse IgG Fcγ (Jackson Labs,1:5000 dilution in PBS/Tween), 150 μl/well, and incubated for 1 hour atroom temperature. o-phenylenediamine (OPD) peroxidase substrate (Sigma)was made and added to the washed plates, 150 μl/well and incubated fortwenty minutes at room temperature. The reaction was stopped by adding2N HCl, 50 μl/well. Absorbances at 490 nm were measured by a platereader from Molecular Devices.

Cells from wells identified as positive by this method were subcloned bylimiting dilution two times. For each subcloning, mAb-secreting cloneswere identified using the recombinant Ly1448P protein ELISA assay. 4cloned mABs were isolated and isotyped: 7A9, IgG1; 11E1, IgG1; 18B9,IgG2a; 18H10, IgG1. The specific epitope recognized by these mAbs wasdetermined using synthetic peptides encoding different segments of theLy1448P protein. This ELISA was performed as described above except thatthe different peptides were used to coat the microtiter wells and eachmAb was tested against each peptide separately in order to determine thelocation of the epitope. Three of the mAbs, 7A9, 11E1, and 18B9 bound toa peptide spaning the C-terminal 25 amino acid segment of Ly1448P asrepresented by amino acids 343-367 of SEQ ID NO:11,050. The 18H10 mAbbound to a 25 amino acid segment of Ly1448P which begins 31 amino acidsN-terminal to the start of the C-terminal peptide and is represented byamino acids 313-336 of SEQ ID NO:11,050. Thus all mAbs were mapped towithin the C-terminal 55 amino acid segment of Ly1448P.

5.11 Example 11 Ly1448 Protein is a Cell Surface Protein

This example illustrates that the Ly1448P protein encoded by SEQ IDNO:9600 is a cell surface protein.

The nucleotide sequence encoding Ly1448P was sub-cloned in to themammalian expression vector pCEP4 (Invitrogen) as follows. The portionof SEQ ID NO:9600 encoding a portion of the Ly1448P protein was PCRamplified using a sense amplimer comprising nucleotides 777-800 of SEQID NO:9600 and additional nucleotides on the 5′ end which contained aHindIII restriction enzyme site and an anti-sense primer comprisingnucleotides 1544-1570 of SEQ ID NO:9600 and additional nucleotides onthe 3′end which contained a NotI restriction enzyme site. The PCRproduct was cloned into the pCR-Blunt vector (Invitrogen), andsequenced. DNA from a pCR-Blunt clone with the correct sequence wasdigested with HindIII and NotI to release the Ly1448P cDNA insert andsubcloned into the mammalian expression vector pCEP4 (Invitrogen) whichhad been previously modified to contain a c-terminal FLAG epitope tag(Sigma; amino acid sequence DYKDDDDK, SEQ ID NO:11,290). The resultingrecombinant vector Ly1448P-FLAG/pCEP encoded a Ly1448P-FLAG fusionprotein.

To determine if the recombinant expression vector Ly1448P-FLAG/pCEPwould express protein, the recombinant vector was transientlytransfected into HEK293 cells using Lipofectamine 2000 according tomanufacturers instructions (GibcoBRL). Separately, HEK293 cells werealso transfected with a control vector containing a recombinant FLAGconstruct and non-recombinant pCEP4 vector. Transfected cells werecultured for 48 hours and then whole cell lysates were prepared. Lysateswere run on a polyacrylamide gel, and the gel was electroblotted ontonitrocellulose membrane using standard techniques (Ausubel et al.,). Theelectroblot was probed with a mouse anti-FLAG monoclonal antibody andbinding of the mouse mAb was detected with a biotinylated rabbitanti-mouse IgG and developed with avidin-HRP and ECL reagent. Anti-FLAGantibodies recognized a Ly1448P-FLAG fusion protein of the expectedmolecular weight.

To determine if recombinant Ly1448P protein was present on the cellsurface, HEK293 cells transiently transfected with Ly1448P-FLAG/pCEP4were biotinylated with Biotin-7-NHS, cells were washed and whole celllysates were generated in TRITON X-100 lysis buffer. Lysates wereimmunoprecipitated with anti-FLAG Sepharose and the resultingimmunoprecipitated material was run on a polyacrylamide gel. The gel waselectroblotted onto nitrocellulose membrane and the blot was developedwith avidin-HRP and ECL reagent. The data show that a proteinprecipitable with anti-FLAG sepharose of the expected molecular weightfor a Ly1448P-FLAG fusion protein was biotinylated in a whole cell assayindicating that Ly1448P is found on the cell surface.

5.12 Example 12 Ly1448P is Expressed on the Cell Surface of B CellLeukemias

This example illustrates that Ly1448P protein is detected on the surfaceof an acute lymphoblastic leukemia cell line SUP-B15 (ATCC Cat #CRL-1929) using polyclonal antibodies described in Example 10.

SUP-B15 cells were cultured according to the suppliers recommendation.For FACS analysis, SUP-B15 and human fibroblasts were collected andwashed with ice cold staining buffer (PBS+1% BSA+Sodium Azide). Next,the cells were resuspended in 50 ul staining buffer and incubated for 30minutes on ice with 75 ul of rabbit anti-Ly1448P recombinant seradiluted 1:500 (see Example 10). Pre-bleed sera was used at the sameconcentration as a negative control. The cells were washed 3 times withstaining buffer and then incubated with a 1:100 dilution of a goatanti-rabbit Ig(H+ L)-FITC reagent (Southern Biotechnology) for 30minutes on ice. Following 3 washes, the cells were resuspended instaining buffer containing Propidium Iodide (PI), a vital stain thatallows for identification of permeable cells, and analyzed by FACS.

The results of the FACS analysis show that the anti-Ly1448P polyclonalantisera was able to recognize and bind to native Ly1448P normallyexpressed on the surface of SUP-B15 cells. Pre-immunization (pre-bleed)antisera obtained from each rabbit used to produce anti-Ly1448P antiseradid not bind to the SUP-B15 cells. Similarly, anti-Ly1448P antisera didnot bind to control human fibroblast cells. These results show that thepolyclonal antisera generated against Ly1448P recombinant protein bindsto Ly1448P naturally expressed on tumor cells such as SUP-B15 and theseantibodies can be used for diagnosis and treatment of B-cellmalignancies such as lymphomas, myelomas and B-cell leukemias such asCLL and ALL.

5.13 Example 13 Novel Splice Junctions Associated with Ly1448P SpliceVariants

This example illustrates the identification of 11 novel nucleotidesequences and 11 novel polypeptide sequences useful for differentiatingamong the multiple splice variants of Ly1448P identified in Example 9.The novel polypeptide junctions contain epitopes that are unique.Antibodies specific for each epitope are useful for determining whichsplice form is expressed in a tumor or tissue sample of interest. Thenucleotide sequences disclosed are useful as probes for determiningwhich splice variant is being expressed in a tumor or tissue sample ofinterest.

Novel junction #1 is identified in splice forms Ly1448P b, d, f, h, i,k, m, and o and shown diagramatically in FIG. 9. Novel junction #1 isgenerated from a splice donor in exon 9 and a splice acceptor in exon(ag)11. Exon 10 is eliminated in this splice variant of Ly1448P. Ly1448Pnovel junction#1 40mer is a polypeptide (SEQ ID NO:11,186) containingthe 20 amino acids either side of the novel protein junction andcontains a novel linear epitope that is recognized by an antibody thatis specific to this polypeptide junction and splice forms that encodeit. Ly1448P novel junction#1 30mer is a shorter polypeptide (SEQ IDNO:11,187) containing the 15 amino acids either side of the same novelprotein junction and contains a more defined novel linear epitope thatis recognized by an antibody that is specific to this junction andsplice forms that encode it. The nucleotide sequence encoding these twopolypeptides are identified in SEQ ID NOs: 11,178 and 11,179respectively.

Novel junction #2 is identified in splice forms Ly1448P c, d, g, h, k,1, o, and p shown diagramatically in FIG. 9. Novel junction #2 isgenerated from a splice donor at the end of exon 11 and a spliceacceptor in exon 13. Exon 12 is eliminated in this splice variant ofLy1448P. Ly1448P novel junction#2 40mer is a polypeptide (SEQ IDNO:11,188) containing the 20 amino acids on either side of the novelprotein junction and contains a novel linear epitope that is recognizedby an antibody that is specific to this polypeptide junction and spliceforms that encode it. Ly1448P novel junction#2 30mer is a shorterpolypeptide (SEQ ID NO:11,189) containing the 15 amino acids either sideof the same novel protein junction and contains a more defined novellinear epitope that is recognized by an antibody that is specific tothis junction and splice forms that encode it. The nucleotide sequenceencoding these two polypeptides are identified in SEQ ID NOs: 11,180 and11,181 respectively.

Novel junction #3 is identified in splice forms Ly1448P e, f, g, h, m,n, o, p, and s, shown diagramatically in FIG. 9. Novel junction #3 isgenerated by a failure to utilize a splice doner present in exon 13resulting in a read through of the splice donor adjacent to thetransmembrane domain and the synthesis of novel peptides encoded by theremainder of exon 13. Ly1448P novel junction#3 38mer is a polypeptide(SEQ ID NO:11,190) containing the 20 amino acids to the amino side ofthe exon 13 splice donor and the 18 amino acids to the carboxy side ofthe exon 13 splice donor. This polypeptide contains a novel linearepitope that is recognized by an antibody that is specific to thispolypeptide junction and splice forms that encode it. Ly1448P noveljunction#3 30mer is a shorter polypeptide (SEQ ID NO:11,191) containingthe 15 amino acids on either side of the same exon 13 splice donor andcontains a more defined novel linear epitope that is recognized by anantibody that is specific to this junction and splice forms that encodeit. The nucleotide sequence encoding these two polypeptides areidentified as SEQ ID NOs: 11,182 and 11,183 respectively.

Novel junction #4 is identified in splice form Ly1448P q1 and showndiagramatically in FIG. 9. Novel junction #4 is generated from a novelsplice doner in exon 10(gt) and a splice acceptor in exon (ag)11. Aportion of exon 10 is eliminated and 4 novel amino acids encoded by exon(ag)11 are incorporated in this splice variant. The novel amino acidsresult from the use of a different reading frame for that portion exon(ag)11 contained in this splice variant. Ly1448P novel junction#4 24meris a polypeptide (SEQ ID NO:11,192) containing the 20 amino acids to theamino side of the novel protein junction and the 4 amino acids to thecarboxy side of the novel protein junction. This polypeptide contains anovel linear epitope that is recognized by an antibody that is specificto this polypeptide junction and splice forms that encode it. Ly1448Pnovel junction#4 19mer is a shorter polypeptide (SEQ ID NO: 11,193)containing the 15 amino acids to the amino side of of the novel proteinjunction and the 4 amino acids to the carboxy side of the novel proteinjunction. This polypeptide contains a novel linear epitope that isrecognized by an antibody that is specific to this polypeptide junctionand splice forms that encode it. The nucleotide sequence encoding thesetwo polypeptides is disclosed in SEQ ID NOs: 11,184 and 11,185respectively.

Novel junction #5 is identified in splice forms Ly1448P r and s showndiagramatically in FIG. 9. Novel junction #5 is generated by atranscript containing all of exon 12. Translation of the entire exon 12generates a peptide containing 42 amino acids that are not contained inother Ly1448P variants. The nucleotide sequence encoding the polypeptidesequence unique to Ly1448P splice variants r and s and the encodedpeptide are disclosed in SEQ ID NOs:11,206 and 11,209 respectively.Ly1448P #5 plus20mer is a polypeptide (SEQ ID NO:11,211) containing theunique Ly1448P variants r and s polypeptide and the 20 amino acidsadjacent to the novel peptide encoded by exon 13. This polypeptidecontains novel linear epitopes that are recognized by antibodies thatare specific to this polypeptide and the unique junction encoded byLy1448P splice forms r and s. Ly1448P #5 plus15mer is a shorterpolypeptide (SEQ ID NO: 11,210) containing the unique Ly1448P variants rand s polypeptide and the 15 amino acids adjacent to the novel peptideencoded by exon 13. This polypeptide contains more defined novel linearepitopes that are recognized by antibodies that are specific to Ly1448Pvariants r and s and this junction encoded by exon 13. The nucleotidesequence encoding these two polypeptides is included as SEQ IDNOs:11,208 and 11,207 respectively. Antibodies raised against the uniqueLy1448P variants r and s polypeptides described Ly1448P #5 plus15mer andLy1448P #5 plus20mer are specific to Ly1448P variants r and s. SPAP1c isa variant of Ly1448P variants r and s. SPAP1c contains the same aminoterminal 135 amino acids as Ly1448P variants r and s (also know asSPAP1a and 1b) but contains a unique 9 amino acid carboxy terminus (SEQID NO:11,293). Antibodies raised against this 9 amino acid region wouldbe able to differentiate between SPAP1c and Ly1448P variants r and s.All of these antibodies are used in the diagnosis, monitoring andtherapeutic treatment of hematologic malignancies in general and CLLspecifically.

Antibodies that recognize these epitopes described above specificallyrecognize splice variants of Ly1448P that encode these epitopes anddifferentiate from splice variants that do not contain them. Moreover,inclusion of different and novel polypeptide junctions, as a result ofdifferential splicing result in different secondary, tertiary structuresof the molecules that contain them. Antibodies directed toward theLy1448P molecules that occur as a result of differential splicingrecognize these novel splice forms based on their unique and differentsecondary and tertiary structures. Antibodies that recognize thedifferent Ly1448P molecules, based on differences in secondary andtertiary structure that result from the differential splicing, identifyspecific different splice variants of Ly1448P and differentiate betweenthem. Antibodies (naked and conjugated) directed to the specific spliceforms can be used to identify and target these splice forms that may bedifferentially expressed between different hematologic malignancies(including, but not necessarily limited to, different classes of B-cellnon-Hodgkins lymphoma and CLL). Therefore, antibodies directed to one ormore of the Ly1448P splice forms provide for a better diagnostic ortherapeutic antibody in one clinical indication verses another.

The Ly1448P variants r and s polypeptides described contain T-cellepitopes that can be utilized as a T-cell therapeutic vaccine for thetreatment of hematologic malignancies in general and CLL specifically.Polypeptide sequences (9mers) contained within polypeptide sequenceLy1448P #5 plus20mer that are predicted to be bound by different HLAclass I subtypes by the EpiSeek HLA class I binding prediction program(Parker et al., J. Immunol. 152:163-175 (1994)) are disclosed in SEQ IDNOs:11,212-11,287.

5.14 Example 14 Identification of IRTA 1, 2a, 2b, 5 and Ly1448P SpliceVariants in B-Cell Neoplasias

This example illustrates that the cell surface proteins IRTA 1, 2a, 2b,5 and Ly1448P splice variants are expressed in B-Cell neoplasias.Diagnostic and therapeutic antibodies and compounds directed at thesemolecules are useful for treating the neoplasias expressing theseproteins.

Real Time PCR analysis was performed on a panel of cDNAs derived fromCLL patient samples, lymphoma patient samples, normal hematologic cellsand normal tissues. Primers were designed that were specific for each ofthe IRTA or Ly1448P family members. Forward and reverse IRTA1 primersare disclosed in SEQ ID NOs:11,194 and 11,195 respectively. Forward andreverse IRTA 2a primers are disclosed in SEQ ID NOs:11,196 and 11,197respectively. Forward and reverse IRTA 2b primers are disclosed in SEQID NOs:11,198 and 11,199 respectively. Forward and reverse primersamplifying a portion of exon 11 contained in Ly1448P splice variants a-pand q2 are disclosed in SEQ ID NOs:11,200 and 11,201 respectively.Forward and reverse primers amplifying a portion of exon 12 contained inLy1448P splice variants r and s are disclosed in SEQ ID NOs:11,202 and11,203 respectively. Forward and reverse IRTA5 primers are disclosed inSEQ ID NOs:11,204 and 11,205 respectively. Forward and reverse CD20primers are disclosed in SEQ ID NOs: 11,288 and 11,289 respectively.

Real Time PCR was performed as described by Gibson et al., GenomeResearch 6:995-1001 (1996) and Heid et al., Genome Research 6:986-994(1996) using a Perkin Elmer/Applied Biosystems 7700 Prism instrument.Matching primers and fluorescent probes are designed for genes ofinterest, for example, by using the primer express program provided byPerkin Elmer/Applied Biosystems. Optimal concentrations of primers andprobes are determined by routine experimentation, and control (e.g.β-actin) primers and probes are obtained commercially from, for example,Perkin Elmer/Applied Biosystems. Standard curves are generated using theCt values determined in the Real Time PCR, which are related to theinitial cDNA concentration used in the assay. Standard dilutions rangingfrom 10-10⁶ copies of the gene of interest are generally sufficient. Inaddition, a standard curve is generated for the control sequence. Thispermits the standardization of initial RNA content of a tissue sample tothe amount of control for comparison purposes.

Real Time results were expressed as deltaCT (each sample normalized toactin but a gene of interest standard curve was not run to determinecopy# of actin message/pg of RNA) and are therefore values are onlyrelative to each other within a specific experiment/run. Absolute valueswere approximated between experiments by comparing the mean cycles takenfor half maximal amplification. For all experiments/runs actin required20-23 cycles to be amplified. By comparison the CD20 gene, a highlyexpressed B-cell marker, required 21 cycles to be amplified from CD19+sorted cells (13 cells), exon 12 containing Ly1448P splice variantsrequired 26 cycles to be amplified from the same population of cells andexon 11 containing Ly1448P splice variants required 33 cycles.

The results indicate that Ly1448P splice variants r/s containing fulllength exon 12 were overexpressed in 59% (10/17) of CLL samples and hadno detectable expression (0/5) in B-cell Non-Hodgkinslymphoma/Hodgkinssamples at a level comparable or greater than that seen in CD19+ cells.Ly1448P splice variants a-p and q2 containing a portion of exon 11 wereonly expressed in 6% (1/17) of CLL samples but was seen in 100% (5/5) ofB-cell Non-Hodgkins lymphoma/Hodgkins samples at a level comparable orgreater than that seen in CD19+ cells. Thus, over-expression of exon 11containing Ly1448P splice variants is specific to CD19+ cells and B-cellnon-Hodgkins lymphomas/Hodgkins lymphoma samples, whereas overexpressionof full length exon 12 containing Ly1448P splice variants is specific toCD19+ cells and a majority of CLL samples. Both types of splice variantsare expressed in the single follicular lymphoma sample analysed.However, expression of exon 12 containing Ly1448P splice variants isconsiderably higher than the expression of exon 11 containing Ly1448Psplice variant, requiring only 23-25 cycles to be amplified as comparedto 29-31 cycles.

Similarly, IRTA2a/b and IRTA5 also show overexpression in CLL samples ata level comparable to that seen with exon 12 containing Ly1448P splicevariants. IRTA1, in contrast, is overexpressed in lymphoma at lowerlevels similar to those observed for exon 11 containing Ly1448P splicevariants. These RNA expression data indicate that exon 12 containingLy1448P splice variants, IRTA2a/b and IRTA5 are overexpressedspecifically in CLL at levels that are lower than seen for CD20 but atlevels that result in significant protein production. Exon 12 containingLy1448P splice variants, IRTA2a/b and IRTA5 constitute a CLL-specificdiagnostic and therapeutic target. Exon 11 containing Ly1448P splicevariants and IRTA1 are not expressed significantly in CLL but areoverexpressed in lymphoma. Exon 11 containing Ly1448P splice variantsand IRTA1 constitute a lymphoma specific diagnostic and therapeutictarget.

5.15 Example 15 Expression of SPAP1a

This example illustrates the cloning of SPAP1a (Ly1448P variant r) intothe expression vector pCEP4 flag. This disclosure also demonstrates thatmouse immunization with peptides covering the unique amino terminal 42amino acid domain of SPAP1a generates production of sera antibody whichbinds SPAP1 but not irrelevant peptides. These polyclonal serapreferentially bind HEK transfected with the SPAP1a-pCEP4 flag constructover an empty pCEP4 construct, thus demonstrating surface expression ofthe SPAP-1a molecule. Finally, this example illustrates that both thesera, as well as an anti-flag antibody, identify in western blots anoverexpressed 32 kd band in lysates of SPAP1a-pCEP4 flag-transfected HEKcells which is not present in lysates of pCEP4 (emptyvector)-transfected HEK cells.

Expressing SPAP1a in HEK cells revealed that this antigen can beexpressed in mammalian cells on the surface and in the total cell.Positive sera titers by ELISA of SPAP1a peptide immunized micedemonstrated the immunogenicity of the antigen.

Cells expressing SPAP1a may be used as an immunogen for therapeuticmonoclonal antibody generation or for use in the generation of SPAP1aspecific CTL.

Full length SPAP1a cDNA template was generated by rt PCR. SPAP1a wasPCR-amplified using primers encoding Hind III restriction site 5′ of theORF (gtaagcttaccatgtgggaatggaaaatatgcaac) (SEQ ID NO:11,294) and a Not Isite 3′ of the ORF (ggtagcggccgctgatttcttcacagaagagtagatgac) (SEQ IDNO:11,295). This PCR product was cloned into a TOPO blunt shuttle vector(Invitrogen) containing Hind III and Not I cloning sites and akanamycin-resistance gene. This material was used to transformchemically competent E. coli, which were plated to kanamycin-containingagarose plates. Following an overnight incubation, a select clone wasgrown at 37° C. with shaking in 2×YT media and kanamycin. The plasmidDNA was isolated. The plasmid DNA and pCEP4 vector containing a 5″ HindIII cloning site, a flag tag epitope 3′ of the Not I cloning site asdescribed above, and an ampicillin-resistance gene were both digestedwith Hind III and Not I. Following gel purification, the SPAP1a insertand pCEP4 flag vector were ligated. Chemically competent bacteria weretransformed with this product and plated to carbenicillin containingagarose plates. Following an overnight incubation, a select clone wasgrown at 37° C. with shaking in 2×YT media and carbenecillin. The DNAplasmid were sequenced and a clone was selected for validation.

An ELISA was performed to determine titers of Anti-SPAP1a antibodycontained in the sera of four mice immunized two times with peptidesspanning the unique amino terminus of SPAP1a. Peptide 1 contained aminoacids 1-25 of SEQ ID NO:11,057, and peptide 2 contained amino acids20-42 of SEQ ID NO:11,057. Plates were coated with peptides (10micrograms/ml) of SPAP1a peptides or irrelevant peptides. Plates wereblocked with 1% BSA/PBS/Tween 20 (Sigma). Following 6 plate washes,serum dilutions were plated in blocking buffer and incubated. Plateswere washed, and then anti-mouse-HRP antibody (1:10,000) was plated.Plates were incubated, then washed again. Plates were developed withperoxidase substrate, then quenched with 1N H₂SO₄, and read forA450-A570. ELISA results demonstrated that sera from mice immunized withSPAP1a peptides 1 and 2 have reactivity to SPAP1a peptides 1 and 2 butnot irrelevant peptide at a 1:102400 dilution.

SPAP1a expression was validated by transient transfection of HEK cellswith SPAP1a-pCEP4-flag recombinant vector described above, followed byFACS staining with sera of SPAP1a peptide immunized mice, or westernblot probing with the same sera or anti-flag antibody. SPAP1a-pCEP4 flagwas validated by transfection of HEK with the construct, followed byflow cytometry to identify surface expression and western blot analysisto identify total expression. Transfection of HEK was performed with twoconstructs: SPAP1a-pCEP4 flag and pCEP4, which contained neither SPAP1anor a flag epitope. 2.5 micrograms of DNA and 5 microliters ofLipofectamine 2000 were put into separate tubes containing 125microliters of Optimem (Gibco) and incubated 5 minutes at roomtemperature. Then contents were combined and incubated an additional 20minutes prior to the addition to 90% confluent HEK in a 6-well plate.Following two days of culture at 37° C., samples were harvested andprepared for flow cytometric analysis and western blotting. Flowcytometry samples (100000 cells/well) were washed and stained inPBS+0.5% BSA+10 micrograms/ml anti-human immunoglobulin. Samples werestained with dilutions of sera of SPAP1a peptide-immunized mouse #4 orsera of a mouse-immunized with an unrelated antigen (irrelevant sera)for 30 minutes on ice. Following three washes, samples were stained witha secondary phycoerythrin-conjugated goat anti-mouse immunoglobulin(human immunoglobulin-adsorbed) for 30 minutes on ice. Samples werewashed three times before the addition of Pharmingen Via-Probe for thestaining of permeabilized cell DNA. Flow cytometry samples were gated soas to collect only the large, dye-impermeable (viable) cells foranalysis of phycoerythrin staining. The FACS analysis demonstrate thatsera of SPAP1a-peptide immunized mouse #4 preferentially bindsSPAP1a-pCEP4 flag-transfected cells over pCEP4-transfected cells, whileirrelevant mouse sera does not. West blot samples were prepared bywashing the harvested transfected cells three times in PBS before lysingthe pellet in media containing Triton X-100 and protease inhibitors.Lysates were centrifuged at 14000 g and the protein containingsupernates were harvested. Samples containing supernatants of equivalentnumbers of cells received 2-mercaptoethanol and were boiled for 10miniutes. Samples were loaded onto and SDS-PAGE gel, run 200V×1 hr, andtransferred to a membrane at 25V for 30 minutes. The blot was blockedwith TBS-0.1% Tween 20+10% milk for 45 minutes. It was cut in half andprobed with primary reagents in TBS-Tween 20+1% milk for 1 hour. Onehalf was probed with a mouse anti-flag antibody (3.33 micrograms/ml),the other half with sera of SPAP1a-peptide-immunized mouse #4 (1:100dilution). Following three washes, the blots were probed with donkeyanti-mouse immunoglobulin (H+L)-HRP in TBS-Tween-20-1% milk for 1 hour,and washed 3×. The blot was reassembled and developed withchemiluminescent reagent. SPAP1a was expressed as a dominant band at 32kd, and is apparent in lanes of SPAP1a-flag/HEK probed by either theanti-flag or the anti-SPAP1a mouse sera, but not in the pCEP4/HEK lanes.

6. REFERENCES

The following references, to the extent that they provide exemplaryprocedural or other details supplementary to those set forth herein, arespecifically incorporated herein by reference.

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All of the compositions and methods disclosed and claimed herein can bemade and executed without undue experimentation in light of the presentdisclosure. While the compositions and methods of this invention havebeen described in terms of preferred embodiments, it will be apparent tothose of skill in the art that variations may be applied to thecomposition, methods and in the steps or in the sequence of steps of themethod described herein without departing from the concept, spirit andscope of the invention. More specifically, it will be apparent thatcertain agents which are both chemically and physiologically related maybe substituted for the agents described herein while the same or similarresults would be achieved. All such similar substitutes andmodifications apparent to those skilled in the art are deemed to bewithin the spirit, scope and concept of the invention as defined by theappended claims. All publications, patents, and patent applicationscited herein are hereby incorporated by reference in their entirety forall purposes. Accordingly, the exclusive rights sought to be patentedare as described in the claims below:

1-21. (canceled)
 22. An isolated monoclonal antibody that specificallybinds to a polypeptide encoded by a nucleic acid sequence set forth inSEQ ID NO:
 9601. 23. A nucleic acid encoding the monoclonal antibody ofclaim
 22. 24. The monoclonal antibody of claim 22, wherein saidmonoclonal antibody specifically binds to amino acids 1-42 of thepolypeptide encoded by a nucleic acid sequence set forth in SEQ ID NO:9601.
 25. A pharmaceutical composition comprising a monoclonal antibodyaccording to claim 22 and a pharmaceutically acceptable carrier.
 26. Themonoclonal antibody of claim 22, wherein said antibody is a chimericantibody.
 27. The monoclonal antibody of claim 22, wherein said antibodyis a Fab fragment.
 28. The monoclonal antibody of claim 22, wherein saidantibody is a Fv fragment.
 29. The monoclonal antibody of claim 22,wherein said antibody is a scFv.
 30. The monoclonal antibody of claim 22further comprising a reporter group.
 31. The monoclonal antibody ofclaim 22 further comprising a therapeutic moiety.
 32. The monoclonalantibody of claim 31, wherein the therapeutic moiety is a radionuclide.33. A pharmaceutical composition comprising a monoclonal antibody ofclaim 22, and a pharmaceutically acceptable carrier.
 34. Use of aneffective amount of a pharmaceutical composition comprising an isolatedmonoclonal antibody that specifically binds to a polypeptide encoded bya nucleic acid sequence set forth in SEQ ID NO: 9601 and apharmaceutically acceptable carrier in the manufacture of a medicamentfor treatment of a hematological malignancy in a mammalian subject. 35.The method of claim 34, wherein the hematological malignancy isassociated with overexpression of SPAP.
 36. The method of claim 34,wherein the hematological malignancy is selected from the groupconsisting of lymphoma, B cell leukemia, multiple myeloma, andcombinations thereof.
 37. The method of claim 34, wherein thehematological malignancy is chronic lymphocytic leukemia.
 38. The methodof claim 34, wherein the mammalian subject is a human.
 39. The method ofclaim 34, wherein the administration is intravenous.
 40. A method forthe detection of a hematological malignancy in a patient, said methodcomprising: (a) contacting a biological sample from the patient with amonoclonal antibody that specifically binds to a polypeptide encoded bya nucleic acid sequence set forth in SEQ ID NO: 9601, whereby saidmonoclonal antibody forms a complex with a polypeptide encoded by SEQ IDNO:9601 (b) detecting the amount of said complex, thereby detectingcancer in said patient.
 41. The method of claim 40, wherein thehematological malignancy is selected from the group consisting of:lymphoma, B cell leukemia, and multiple myeloma, and combinationsthereof.
 42. The method of claim 40, wherein the hematologicalmalignancy is chronic lymphocytic leukemia
 43. A kit for detecting ahematological malignancy cell, said kit comprising: a monoclonalantibody that specifically binds to a polypeptide encoded by a nucleicacid sequence set forth in SEQ ID NO: 9601; and instructions for use.